Practical Applications of Formaldehyde and Organic Acids in Control

King Industries, Inc. High Performance Products for Coatings, Inks, Adhesives andSealantsNACURE® & K-CURE®Acid & Blocked Acid CatalystsK-KAT®Non-tin Catalysts for UrethanesNACURE® SUPER CATALYSTSCationic Cure of EpoxiesK-FLEX®Resin Modifiers & Reactive DiluentsK-STAY®Rheology ModifiersK-SPERSE®Wetting & Dispersing AdditivesNACORR®Rust & Corrosion InhibitorsDISPARLON®Thixotropes & Surface Control AdditivesTechnology OverviewSince 1932, King Industries has been supplying specialty chemical products to a variety of industries that are performance driven with ever changing requirements. This is especially true for the coatings, inks, adhesivesand sealant markets, the audience for this product guide. While the brochure covers our standard products,this overview has been designed to give you a summary of our areas of technical expertise and to urge youto contact us if you feel we may be of assistance for your specific product needs.CATALYSISWith over four decades of experience in catalysis, King offers the industry’s broadest spectrum of catalysts including:• Acid and blocked acid (latent) catalysts for amino thermoset systems• Non-tin, mercury-free catalysts for urethanes, foams and cast elastomers• Latent and super acid catalysts for the cationic cure of epoxies• Hydrophobic catalysts for the moisture cure of siloxane functional polymers• Powder catalysts for uretdione crosslinked powder coatings and caprolactam blocked isocyanate powder systemsCORROSION INHIBITIONKing offers ferrous and non-ferrous protection for a wide variety of metals and systems based on threeunique platforms:• Modified trialzole compounds• Amino acid derivativesDISPERSANT TECHNOLOGYWhether organic or inorganic pigment/fillers, King offers a variety of dispersant technologies including:• Solvent free polymeric wetting and dispersing agents for solventless and epoxy systems• Sulfonate based dispersants for non-aqueous, solvent-free and powder systemsRESIN MODIFIERS/REACTIVE DILUENTS• Unique polyester polyols based upon low molecular weight, linear, saturated aliphatic structures with pendent hydroxyl groups• Novel, low molecular weight diols with an all urethane backbone• Acetoacetate functional reactive diluentsRHEOLOGY MODIFIERS• Unique sulfonate based modifiers for non-aqueous systems• Polyamide based thixotropes• Hydrophobically modified ethoxylated urethane thickeners for waterborne systemsSURFACE CONTROL ADDITIVESIn addition to King’s internally developed products, the Disparlon® product line represents over 25 years of a technology alliance with Kusumoto Chemical Ltd. of Japan. The Disparlon line offers a broad range of level-ing, defoaming, anti-popping and anti-cratering additives for aqueous, solvent, solventless, UV and powder systems.© All materials copyrighted 2006, King Industries, Inc., Norwalk, CT, USAPRODUCT LINESSYSTEMHigh SolidsWaterborneConventionalPowderUVSECTION I - CATALYSTSNACURE® & K-CURE®ACID & BLOCKED ACID CATALYSTS 3 K-KAT® NON-TIN CATALYSTS FOR URETHANES9NACURE® SUPER CATALYSTS LATENT CURE OF EPOXIES13SECTION II - RESIN MODIFIERS/REACTIVE DILUENTSK-FLEX®SPECIALTY PRODUCTS 15 K-FLEX®POLYESTER POLYOLS 17K-FLEX®URETHANE DIOLS21SECTION III - SPECIALTY ADDITIVESK-STAY®RHEOLOGY MODIFIERS23 NACORR®RUST & CORROSION INHIBITORS 25 K-SPERSE®WETTING & DISPERSING ADDITIVES 28 DISPARLON® THIXOTROPES30 DISPARLON® DEFOAMERS & ANTI-POPPING AGENTS35 DISPARLON® DISPERSANTS,ANTI-FLOOD & ANTI-FLOAT AGENTS 36DISPARLON® LEVELING & ANTI-CRATERING ADDITIVES37CONTACT INFORMATION: PAGEAPPLICATION CHART Centerfold Table of Contents and System Reference ChartWorld Headquarters King Industries, Inc.Science Road, CT 06852 USA(800) 431-7900 or (203) 866-5551 (203) 866-1268coatings@European Sales Office King International EuropeNoordkade 64, 2741 EZ Waddinxveen The Netherlands+31 182-631360 +31 182-621002 info@kingintl.nlNACURE ® & K-CURE ®Acid & Blocked Acid CatalystsCatalyst By Acid Type Acid Type Acid Catalysts Blocked Catalysts NACURE 155 NACURE X49-110 NACURE 3525NACURE 3327 NACURE 3483 NACURE 1051 NACURE 1323 NACURE 1419NACURE 1557 NACURE 1953NACURE 5076 NACURE 5225NACURE 5414NACURE 5528 NACURE 5925 K-CURE 1040 K-CURE 1040W NACURE 4054 NACURE XC-C207 NACURE 4167 NACURE XP-297 NACURE 4575 NACURE 2107 NACURE 2500 NACURE 2501NACURE 2522 NACURE 2530 NACURE 2547 NACURE 2558AAP & PAPAlkyl Acid Phosphate Phenyl Acid Phosphate Why Use Catalysts? Today’s need for high solids and waterborne coatings requires greater use of high reactivity, lowviscosity resins and crosslinkers. Conversion ofthese systems into tough, chemically resistant, high performance coatings at reduced cure temperatures can be accomplished with the use of a catalyst. Acrylics, alkyds, epoxies and polyesters with reactive functional groups, such as hydroxyl, carbamate or amide can be reacted with melamine, urea and benzoguanamine crosslinkers. Selection of the proper catalyst can facilitate the crosslinking reaction resulting in the following benefits:• Shorter cure schedules• Lower cure temperatures for thermoset highsolids and waterborne coatings• Improved hardness, gloss, humidityand corrosion resistance • Improved mechanical propertiesKing Industries continues to develop catalysts to meet the ever expanding needs of a rapidly changing market.Free Acid Or Latent Catalyst? While acid catalysts provide the fastest cure and lower curing temperatures, blocked catalysts aretypically chosen for systems requiring greater package stability. In addition, troublesome catalyst-pigment interaction can be reduced oreliminated.As can be seen in the table which follows, King’s catalyst line is based upon a variety of acids shownin their structural form. The middle column denotesthe free acid versions while the far right columnshows amine blocked or covalently bondedderivatives for applications requiring extended package stability.Catalyst SelectionThe first thing to consider when selecting a catalyst is what type of crosslinking agent is being used. High solids and waterborne coatings are typicallyformulated with monomeric crosslinkers such as hexa(methoxymethyl)melamine (HMMM) or mixed ether melamine; reaction of these crosslinkers with hydroxy or carbamate functional groups is best achieved with strong acid catalysts like DNNDSA or p-TSA.N A C U R E & K -C U R E A C I D & B L O C K E D A C I D C A T A L Y S T SSO 3H C 9H19HO 3S H 19C9DNNDSAC 9H 19SO 3H H 19C 9DNNSA SO 3HC 12H 25DDBSA CH3SO 3HpTSAMore reactive crosslinkers, which are more polymeric but contain high levels of -NH groups, respond better to a weaker acid such as acid phosphates or low dosages of amine blocked sulfonic acids. The chemical structure of the catalyst, as well as the quantity used, can have a profound impact on such film properties as adhesion, corrosion resistance, flexibility and impact resistance. These differences are apparent not only among different acid types but also among different products within the same chemical family. The table that follows matches the type of crosslink-ing agent and the acid catalyst most suitable for each class.Formulating ConsiderationspH Range - Both fully and partially alkylated amino resins are reactive under acidic pH conditions andrelatively stable in the neutral range. To acceleratethe reaction between binder resin and amino crosslinker, it is necessary to reduce the pH of thesystem through the addition of an acid catalyst. Forfully alkylated melamines, a pH of 3 or lower isrequired to induce cure. Partially alkylated melamines of the high imino type will react in the pH range of 3 to 5.Blocking the acid catalyst with an amine will effectively raise the pH, inhibiting the cure at low temperatures and allowing the formulation of stable one package systems. Amine blocked catalysts canCrosslinking Agent General Acid Category Acid Types Fully alkylated monomeric M/F resins: Fully methylated Fully butylated Mixed ethers Urea formaldehyde resins Benzoguanamine resinsGlycoluril resinsStrong Acids pKa<1 Highly alkylated, high imino M/F resins Partially alkylated poly-meric M/F resinsWeak Acids pKa 1-3Metal SaltsCarboxylic Acid PhosphatesRELATIVE ACID STRENGTH:p-TSA>DNNDSA>DDBSA>DNNSA>Phosphates>CarboxylatesP-TSA DNNDSADDBSADNNSA be prepared at virtually any pH, but usually the best combination of cure and package stability is obtained in the 6.5-7.5 range. Cure Schedule and Temperature Generally, the time and temperature conditions of cure can prescribe the correct catalyst for the application. Strong acids with typical pKa strengths of approximately 0.5-0.7 should give equivalent rates of cure at equal molar concentrations of the acid group. Amine neutralized or polymeric blocked catalysts will demand higher temperatures for full activation, and the pKa of the amine and type of polymer attached to the acid will also influence therate of reactivity. On the next page a table can be found that providesthe various cure profiles for acid catalysts basedupon a 30 minute cure schedule for a typicalResin/HMMM (75/25 ratio) coating. Likewise, priorto the blocked catalyst descriptions, a graph showscure profiles for blocked catalysts under the same conditions.In both cases, the data should be viewed as astarting point and a ladder study should be conducted to optimize the formulation.Quick Formulating Tips Do not over catalyze. Using too much catalyst canbe a costly mistake and one that can cause film properties to suffer significantly.As with any component in a coating, the level, method and order of addition may mean the difference between formulation success or failure. When incorporating catalysts, the following factors should be considered: method of mixing, solvents used, pigments used, pH sensitivity of the resins, temperature at time of addition, substrate and stability/pot life requirements. While some general recommendations can be found in the product description charts, feel free to contactKing’s Technical Service Department at (800) 431-7900 or by email coatings@ for assistance in selecting the proper catalyst for your particular application. N A C U R E & K -C U R E A C I D & B L O C K E D A C I D C A T A L Y S T S4NACURE ® & K-CURE ® Acid CatalystsPRODUCT Acid Type Volatile % Active Acid # lbs./gal. Gardner Color Minimum Cure*Attributes/UsesNACURE 155DNNDSA Isobutanol 55 112-116 8.16 12 max. RT General purpose catalyst. Excellent water, detergent and salt spray resistance.NACURE 1051 DNNSA2-Butoxyethanol50 60-64 8.16 N/A 125°C Best water and corrosion resistance. Recommended for high temperature applications on metal. NACURE 5076 DDBSA Isopropanol 70 130-140 8.27 4 RT Complies with FDA 21 CFR, Sec. 175.300 (b) (3) xiii (a&b)NACURE 4054 AAP Isobutanol50155-165 7.49 1 110°C Weak acid for high NH/polymeric melamines and phenolic crosslinkers.K-CURE 1040 p-TSA Isopropanol 40 130-140 8.25 1 RT Highest gloss. Fastest cure. Excellent weathering and exterior durability.K-CURE 1040W p-TSA Water 40 130-140 9.40 2 RT As above, non-flammable for waterborne applications.K-CURE 129B Mixed AcidsMethanol/n-Butanol50 200-210 8.90 1 RT Fastest cure.Wood and paper coatings.NACURE XC-C207Alkyl Acid Phosphate10065011.8180˚CBroad solubility and excellent ad-hesion, Good package stabilityRT= Room Temperature, cures are possible at catalyst levels of 4-10% *30 minute cure schedule – Resin/Urea (60/40 ratio)NACURE 4046 Phosphate Xylene/Butanol 17 100-112 7.60 2 80°C Complies with FDA 21 CFR, Sec. 175.300 (b) (3) xiii (a&b) Acid Catalysts - Suggested Starting LevelsProduct 70°C90°C110°C125°C150°C175°C200°CNACURE 155 4.7% 2.7% 1.6% 1.2% 0.6% 0.5% 0.3% NACURE 1051 NR NR NR 2.2% 1.4% 0.9% 0.6% NACURE 5076 4.5% 2.5% 1.5% 1.1% 0.7% 0.5% 0.3% NACURE 4054 NR NR 4.0% 2.0% 1.0% NR NR K-CURE 1040/W 4.2% 2.3% 1.4% 1.0% 0.7% 0.4% 0.3% K-CURE 129B 2.6%1.5%0.9%0.7%0.4%0.3%0.2%30 Minute Cure Schedule, catalyst as supplied on Total Resin Solids, Resin/HMMM (75/25 ratio), NR=Not recommendedNACURE 4046 NR 3.0% 2.0% 1.5% 1.0% 0.8% 0.5% NACURE XC-C2072.5% 2.0% 1.0% 0.75% 0.5% 0.3% 0.2%Acid Catalysts - Typical Use LevelsThe chart below can be used as a starting point guideline. The suggested cure schedules are based upon a 30 minute cure for typical Resin/HMMM (75/25 ratio) coatings. The suggested starting levelis the percentage of catalyst based on total resin solids.Once a schedule is established, a ladder study should be conducted to optimize the formulation.N A C U R E & K -C U R E A C I D & B L O C K E D A C I D C A T A L Y S T SNACURE® Blocked Acid CatalystsPRODUCT Acid TypeVolatile%ActivepH lbs./gal. GardnerColorMinimumCure*Attributes/UsesNACURE X49-110 DNNDSAIsobutanolIsopropanol25 6.5 - 7.5 7.55 10 max. 90°CBest overall properties. Excellentwater and corrosion resistance, andadhesion.NACURE 3525 DNNDSAIsobutanolIsopropanol25 7.0 - 8.5 7.65 10 max. 120°CBetter solubility than X49-110,slower curing. Good salt sprayresistance and adhesion.NACURE 3327 DNNDSAIsobutanolIsopropanol25 6.5 - 7.5 7.40 N/A 107°CBetter solubility than other amineblocked DSA catalysts.NACURE 3483 DNNDSAXylene25 N/A 8.20 10 max. 120°CLow conductivity for electrostaticspray. High gloss, reduced pigmentinteraction.NACURE 1323 DNNSAXylene 21 6.8 - 7.5 7.43 N/A 150°CHigh temperature applications.Excellent solubility in aromaticand aliphatic solvents.NACURE 1419DNNSAXylene/MIBK 30 N/A 7.74 N/A 150°CElectrostatic spray. High bakeapplications for water, detergentand salt spray resistance.NACURE 1557DNNSAButanol2-Butoxyethanol25 6.5 - 7.5 7.56 N/A 150°CResolves solvent popping in thickfilms. Excellent humidity anddetergent resistance.NACURE 1953DNNSAButanol2-Butoxyethanol25 6.5 - 6.9 7.48 N/A 150°CHigh bake amino crosslinkedsystems such as coil coatingsand metal decorating.NACURE 5225DDBSAIsopropanol 25 6.0 - 7.0 7.40 2 120°CBest solubility in high solidsenamels. Good solubility inaliphatic solvents.NACURE 5414 DDBSAXylene 25 N/A 8.30 4 130°CPolymeric blocked. Excellentelectrostatic spray (non-aqueous).Good intercoat adhesion.NACURE 5528DDBSAIsopropanol 25 7.0 - 8.0 7.50 2 120°CBroad solubility.Excellent color stability.NACURE 5925DDBSAIsopropanol 25 7.0 - 7.5 7.50 2 120°CComplies with FDA 21 CFR, Sec.175.300 (b) (3) xiii (a&b)NACURE 2107p-TSAIsopropanol 25 8.0 - 9.0 7.57 1 90°CGood metal mark resistance.NACURE 2500p-TSAIsopropanol 26 6.0 - 7.0 8.15 1 80°CLow temperature cure.Excellent stability.NACURE 2501TSAMethanolIsopropanol25 6.0 - 7.2 8.01 1 80°CSlightly higher resistivity than 2500.Better ketone solubility.NACURE 2522p-TSAIsopropanolMethanol25 3.5-3.9 7.85 1 80°CPartially neutralized to provide fastercure and reduce wrinkling at highercuring temperatures.NACURE 2530p-TSAMethanolIsopropanol25 5.7 - 6.5 7.90 1 80°CLow temperature cure.Low tendency to yellow or wrinkle.More Blocked Catalysts Continued On Next PageNACURE&K-CUREACID&BLOCKEDACIDCATALYSTSPRODUCTAcid Type Volatile % Active pHlbs./gal.Gardner ColorMinimum Cure* Attributes/UsesNACURE 2547 p-TSA Water 258.0 - 9.0 9.18 1 107°C Readily soluble in waterborne systems. Solvent-free.NACURE 2558 p-TSA Ethylene Glycol 25 3.5 - 4.5 9.64 1 80°C Excellent control of popping and blistering.NACURE XC-8224Mixed Acids Water258.0 - 9.09.08180°CFast cure response and solubility in waterborne coatings.NACURE 4167 Acid PhosphateIsopropanol Isobutanol25 6.8 - 7.5 7.16 2 80°C Blocked phosphate for high NH/polymeric melamines.NACURE XP-297 Acid PhosphateWater Isopropanol25 6.5 - 7.5 8.20 2 90°C Aqueous systems using high NH/polymeric melamines.NACURE 4575Acid PhosphateMethanol Butanol257.0 - 8.08.302100°CHigh gloss. Superb storage stability with polymeric amino resins.NACURE ® Blocked Acid Catalysts - ContinuedTypical Use Levels - Blocked CatalystsThe chart below can be used to as a starting point reference for blocked catalysts. Cure schedules and use levels are based upon a 30 minute cure for typical Resin/HMMM (75/25 ratio) coatings. The percent of catalyst shown is as supplied on TRS.Once a temperature and use level is established from the chart, a ladder study should be conducted to optimize the formulation.Typical Use Levels for Blocked Catalysts Based On Cure Temperature30 Minute Cure - 75/25 Ratio - Resin/HMMM, % Catalyst (as supplied) on Total Resin Solids100 150 200 250 300 350 400 °F(38) (65) (93) (121) (150) (177) (204) °CBlocked p-TSA - (25% Active) 5%1.6% 0.4%Blocked DDBSA - (25% Active)3%1.3% 0.8%Blocked DNNDSA - (25% Active)6% 2.4% 0.7%Blocked DNNSA - (21% Active)3% 2% 1.1%HydrophobicHydrophilicN A C U R E & K -C U R E A C I D & B L O C K E D A C I D C A T A L Y S T SCatalyst Selection by Applications & PerformanceApplication areas for acid and blocked acid catalysts cover a broad spectrum of end-uses, from medium to high solid solvent-based formulations to water reducible and latex systems. Typical applications are summarized below.The cross reference chart shows you specific performance properties to types of catalysts. As always, our technical service staff can assist you with meeting your particular requirements .ApplicationSolvent SystemsWaterborne SystemsAppliancesNACURE 1323, NACURE X49-110 NACURE 1419, NACURE 3483 NACURE 155, NACURE X49-110NACURE 3525 Automotive BasecoatNACURE 5525, NACURE 2500 NACURE 5528, NACURE 3525NACURE 2500, NACURE 5076 NACURE 2547, NACURE 5528Automotive Topcoat/Clearcoat NACURE 5225, NACURE 2500, 2522, 4054, 5414 & 5528 NACURE 2500, NACURE 5076NACURE 2547, 5528, 5225 and 4167 Can NACURE 5925, NACURE 155NACURE 3483, NACURE 3525 & 4046 NACURE 155, NACURE 5925NACURE 5076 Coil (PCM) NACURE 1051, NACURE 1323 NACURE 1419, NACURE 2107 & 4046NACURE 2500, NACURE 5225NACURE X49-110InksNACURE 155, NACURE 1051K-CURE 1040 NACURE 155 K-CURE 1040W Metal Decorating NACURE 155, K-CURE 1040K-CURE 129B NACURE 155 K-CURE 1040WPaper Coatings K-CURE 1040, K-CURE 129BNACURE 155 K-CURE 1040W, NACURE 155NACURE 2530 Primers NACURE X49-110, NACURE 3525NACURE 1323 NACURE 155, NACURE X49-110NACURE 3525 Wood FinishingNACURE 155, K-CURE 1040K-CURE 129BNACURE 155, K-CURE 1040WNACURE 2530Catalyst Selection by Performance PropertiesPerformance Property Adhesion ▲ ■ ■ Chip Resistance ▲ ■ Corrosion Resistance ▲ ▲ Detergent Resistance ■ ▲ Electrostatic Conductivity ■ ▲ ■ FDA 175.300 ▲ Film Flexibility ▲ ■ Film Hardness ■ ▲ Flow & Leveling ■ ▲ ■ High Gloss▲ ▲ Moisture Resistance ▲ ▲ ■ Package Stability ▲ ■ QUV Resistance▲■KEY: ▲ - Highly Recommended ■ - RecommendedDNNDSA DNNSA DDBSA p-TSA AAP/PAPN A C U R E & K -C U R E A C I D & B L O C K E D A C I D C A T A L Y S T SK-KAT ® Non-Tin Catalysts for PolyurethanesIntroductionAmines and organometallic catalysts are commonlyused as accelerators in the polyol/isocyanate reac-tion to produce polyurethanes. When formulating a polyurethane coating, it has been found that bothperformance and properties can be affected by the choice of catalyst.Amine catalysts are typically used in foam applica-tions. Their catalytic activity accelerates the reactionof aromatic isocyanates with water and alcohols, releasing carbon dioxide. Use of amines in coatingapplications, however, is generally not acceptablebecause of their negative effect on film properties,especially yellowing.King Industries has developed a range ofcatalysts for isocyanate-hydroxyl crosslinking that are based on bismuth, aluminum and zirconium metal chelates and complexes. These K-KAT cata-lysts are not only environmentally more acceptable than organotin compounds, but can offer perform-ance advantages as well.PRODUCT Composition%Non-volatilelbs./gal.Typical Use Levels(% on total resin solids) 2K Blocked NCO Attributes/UsesK-KAT 348 Bismuth Carboxylate7510.00.03-0.1 0.5-2Similar to DBDTL, particularly effective for blocked isocyanates and elastomers K-KAT XC-B221 Bismuth Carboxylate1009.40.03-0.1 0.5-2Improved hydrolytic stability. Especially effective in cationic electro-coatings.K-KAT XC-C227 Bismuth Carboxylate 88 9.3 0.05-0.5 0.5-2Resistant to hydrolysis. Im-proved potlife & high reactivity in forced dried applications K-KAT 4205 Zirconium Chelate 2,4 - PentanedioneN/A 8.11-2 NotRecommendedGood potlife, recommended for ambient cure (not bake/force dry)K-KAT 5218 Aluminum ChelateComplex Reactive Diluent 659.11-2 NotRecommendedExcellent potlife with 2,4, pentanedioneK-KAT 6212 Zirconium Complex Reactive Diluent 95 8.20.3-2 NotRecommended Fast cure, waterborne systems Plural componentK-KAT XK-602 Metal Complex100 Powder 1.0-5.0 Powder CoatingsUretdione crosslinked andcaprolactam blocked isocyanate powder coatings.K-KAT XK-604Organometallic Complex100 10.0 0.1-0.5%100% solids 2K urethanes for cast elastomers. Similar cure profile to mercury catalysts.K-KAT A209 Zirconium Complex Reactive Diluent 35 7.9O0.05-1 NotRecommendedFast cure, waterborne, slow reacting systems Resin synthesisSpecific K-KAT catalysts can be selected to enhance these advantages, including reduced water reaction, improved pot life, faster cure, improved catalysis in cationic electrocoating and reduced hydrolysis of ester groups. Unique Non-tin Catalysts K-KAT 348, XC-B221 and XC-C227 - Bismuth Carboxylate Catalysts Provide properties similar to DBTDL. They are particularly effective in blocked isocyanate and elastomer systems.K-KAT 4205, 6212, and A209 - Zirconium Catalysts can offer fast, selective catalysis of 2K urethane coatings. K-KAT 5218 - Aluminum Chelate Catalyst is used in 2K systems where extended potlife is desired.K-KAT XK-602 - Metal complex designed for powder coatings. K-KAT XK-604 - Mercury Replacement Catalyst based on a proprietary organometallic complexes.K -K A T C A T A L Y S T S F O R U R E T H A N E SK-KAT Products and PerformanceK-KAT 348K-KAT 348 is a bismuth carboxylate which can be used in blocked isocyanate and two component urethane systems offering: • Excellent exterior durability • Non-yellowing characteristics • Excellent gloss retention K-KAT 348 Vs. DBTDL - YellowingA good indicator of resistance to yellowingisb* Color Value where a higher number corresponds to increased yellowing. The graph below demonstrates that similar QUVdurability is obtainable with K-KAT 348 as compared to DBTDL. K-KAT 4205K-KAT 4205 is a liquid zirconium complex which isan effective catalyst for 2K urethane coatings offering: • Fast tack free time • Excellent viscosity stability/pot life • Excellent exterior durability • Use levels at low metal concentrations K-KAT 4205/DBTDL TACK-FREE TIME COMPARISON Polyester/Isocyanate , Ambient Cure, Equal Pot LifeK-KAT 4205 (0.0012% metal) DBTDL (0.0042% metal) 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 HOURSK-KAT 5218K-KAT 5218 can be used in both baked and ambient cured 2K urethane systems offering:• Synergy with pot life extenders such as2,4 - Pentandione• Excellent exterior durabilittyK-KAT 5218/DBTDL POT LIFE COMPARISON 2K Polyester/HDI Trimer, Ambient Cure, Equal Dry TimeK-KAT XC-C227K-KAT XC-C227 offers excellent resistance to hydrolysis compared to conventional bismuth carboxylate catalysts as shown in the photos below.K-KAT XC-C227 Conventional Bismuth CatalystTwo Months Humidity Exposure Open ContainerBismuth carboxylate catalysts will also hydrolyze when diluted with solvents that contain trace amounts of water.K -K A T C A T A L Y S T S F O R U R E T H A N E SQUV STUDY - K-KAT 348/DBTDLAcrylic/Blocked NCO - 30 Minutes/138˚CK-KAT A209 K-KAT A209 is a concentrated version of 6212 that is best suited for slower curing formulations. Typical uses include:• Slow curing formulations such as IPDIcrosslinked coatings• Resin synthesis• NCO terminated prepolymersK -K A T C A T A L Y S T S F O R U R E T H A N E SK-KAT XK-602K-KAT KX-602 was specifically designed to for use in uretdione crosslinked powder coatings to lower cure temperatures while preventing yellowing which is a common problem associated with standard amine based catalysts used in these coatings. These performance characteristics are shown in the table which follows.Polyester/Uretdione Powder CostingTest results show the use of K-KAT XK-602 can lower cure temperatures and even under overbake curing conditions reduce yellowing.TEST RESULTS(Overbake)Control1.25% XK-602 On TRS 5.0% XK-602 On TRSSubstrate Initial Cure Schedule 20 minutes at 200˚ 20 minutes at 170˚C30 minutesat 150°C b*0.12 -0.2 0.43 White index 89.5 90.8 90.8 Yellow index -0.8 -1.7 -0.03 1st Overbake Schedule (∆) 20 minutes at 200˚C 20 minutes at 170˚C 30 minutes at 150°C b*2.5 (2.4) -0.05 (0.20) 0.5 (0.1) White index 79.2 (10.3) 90.1 (0.7) 90.3 (0.5) Yellow index3.7 (4.5) -1.1 (0.6) 0.16 (0.2) 2nd Overbake Schedule (∆) 20 minutes at 200˚C 20 minutes at 170˚C 20 minutes at 200°C b* 3.3 (3.2) 0.09 (0.3) 3.2 (2.8) White index 73.8 (15.7) 89.3 (1.5) 76.9 (13.9) BONDERITE 1000 With the use of an acid scavenger, K-KAT XK-602 has shown to be effective at temperatures as low as 140˚C. K-KAT XK-604 K-KAT XK-604 is based on a blend of proprietaryorganometallic complexes and is highly effectivewhen used to cure cast elastomers, such as 100% solids 2K urethanes. As shown in the profile belowK-KAT XK-604 offers a similar cure profile to mer-cury catalysts without the environmental and toxicity issues.In addition to offering good latency before snap curing, XK-604 can also provide improved post-gel cure compared to other mercury replacement cata-lysts. Gel Cure Profile K-KAT XK-604 to Mercury Catalyst K-KAT XK-604Mercury CatalystK-KAT 6212 K-KAT 6212 is recommended for use in two com-ponent plural gun or in-line mixing applications pro-viding advantages as follows. • Rapid cure response and fast tack free time • Excellent low temperature cure response • High selectivity for - NCO/OH reaction overthe NCO/water reaction K-KAT 6212 must be added to the isocyanate com-ponent.。

Cat. No.Product DescriptionSHH0001-500ML Novec™ 7000 Engineered Fluid SHH0001-1L Novec™ 7000 Engineered Fluid SHH0002-500ML Novec™ 7100 Engineered Fluid SHH0002-1L Novec™ 7100 Engineered Fluid A5597-1GAL Xylene SubstituteA5472-1GAL Formalin Free Tissue Fixative H2779-1L HistoChoice® Clearing AgentH2904-100ML HistoChoice® Tissue FixativeH2904-1L HistoChoice® Tissue Fixative Xylene SubstituteRené J Buesa and Maxim V PeshkovAnnals of Diagnostic Pathology, 13(4), 246-256 (2009) After the hazardous effects of xylene became indisputable in the 1970s, many potential substitutes became available, some with as many if not more hazards. This article discusses the inadequacy of 5 vegetable oils as substitutes, as well as the characteristics of 22 D-limonene-based substitutes, all less effective in their chemical role, some capable of inducing health problems, and costing more than twice as much as xylene. Some of the 35 alkane-based substitutes discussed are effective for tissue processing, less toxic, with a cost about the same as xylene, but are not very effective for dewaxing and other staining tasks. Isopropanol (2-propanol) alone or mixed with molten paraffin is a technically acceptable and cost-effective substitute for xylene for tissue processing, but in this study, we demonstrate that the best clearing agents from the sectioning quality and diagnostic value point of view, with automated or manual protocols, are mixturesof 5:1 and 2:1 isopropanol and mineral oil, followed by undiluted mineral oil, all at 50 degrees C, making thema safer and cheaper substitute than xylene. Using a1.7% dishwasher soap aqueous solution at 90 degreesC to dewax before staining and oven drying the stained sections before coverslipping will eliminate xylene from the staining tasks. Tissue processors retorts and conduits can be dewaxed with a 2% solution of a strong glassware laboratory detergent. These 4 methodologies will make the histology laboratory xylene-free but, due to the natural resistance to change, many histotechs will be reluctantto adopt them if they think that their technical expertise could be jeopardized, and the only way these changes will succeed is if the pathologists, as stewards of the histologylaboratory, commit to their implementation.Go Greener Histology & HematologySigma-Aldrich® is Committed to Bringing Safer Greener Alternatives to Our Customers.• Novec fluids are non-flammable, low toxicity fluidsfor freezing histology samples.• Xylene Substitute is non-toxic version of xylenewhich is compatible with all tissue• Formalin Free Tissue Fixatives is a less toxicalternative to formalin. It is also compatible witha number of applications like PCR, IHC and in-situhybridization• HistoChoice® Clearing Agent is suitable as analternative to toluene and xylene for dewaxing paraffintissue sections• HistoChoice Tissue Fixative is designed for molecularbiology and used for in-situ hybridizationThe life science business of Merck operates asMilliporeSigma in the U.S. and Canada.Formalin Free Tissue FixativesM E Boon and L P KokBiotechnic and Histochemistry, 83(6), 261-277 (2008)The German, F. Blum, introduced formalin as a fixative in 1893. Formalin rapidly became popular for hardening and preserving gross human and animal specimens. As a result, microscopy for diagnostic pathology by combining paraffin embedding and formalin fixation was developed. Alcohol-based fixatives have coagulation of proteins as their main preservative effect. Because there is no cross-linking, immunostaining is not compromised, and DNA and RNA is not damaged. Ethyl alcohol was used by Dutch scientists of the 18th century, but was replaced by the cheaper formalin. Addition of low molecular weight polyethylene glycol (PEG) optimized the coagulant fixative, Kryofix. The polyethylene glycol prevents excessive hardening and enhances the speed of coagulation of proteins. Kryofix was used on a large scale for skin biopsies in Leiden between 1987 and 2001. DNA preservation by the formulated coagulant fixative, BoonFix, is related to the concentration of ethyl alcohol, PEG and acetic acid. BoonFix has been used since 2004 in Leiden for over 40,000 diagnostic skin biopsies and more than 100,000 cervical samples.A literature review and three decades of experience with coagulant, formalin-free fixatives in pathology suggest that when health authorities realizethat formalin invalidates expensive tests, it might eventually be eliminated legislatively from diagnostic pathology. Finally, coagulant fixation is optimal for microwave histoprocessing where ethyl alcohol is followed by isopropanol. HistoChoice® Clearing Agent1. I mmunogenicity and contraceptive potential of three infertility-relevant zona pellucida 2 epitopes in the marsupial brushtail possum (Trichosurus vulpecula).Janine A Duckworth et. alReproduction, 133 (1), 177-186 (2007)In a previous study, three infertility-relevant epitopes of possum ZP2 (Pep12 (amino acids 111-125), Pep31 (amino acids 301-315), and Pep44 (amino acids 431-445)) were identified using sera from possums (Trichosurus vulpecula) immunized with recombinant possum zona pellucida 2 (ZP2) constructs, and a synthetic peptide library of possum ZP2 protein. In this study, the three peptides were conjugated to keyhole limpet hemocyanin and 300 mug of each conjugated peptide were administered subcutaneously to female possums (n = 20 per peptide) in complete Freund's adjuvant. Immunogen doses were repeated 3 and 6 weeks later using incomplete Freund's adjuvant. Control animals were immunized with either phosphate-buffered saline only (n = 10) or 300 mug keyhole limpet hemocyanin (n= 10), administered with the same adjuvants. Serum antibodies from animals immunized against these three epitopes bound to the corresponding possum ZP2 peptides, recombinant possum ZP2 protein constructs, and native zona. Possum fertility was assessed following superovulation and artificial insemination. Peptides Pep12 and Pep31 had no significant effects on fertility parameters (P > 0.05). However, animals immunized with Pep44 had lower egg fertilization rates (immunized 19.5% versus control 60.5%,P < 0.05) and produced significantly fewer embryos than control animals (immunized 0.5 embryos versus control 2.4 embryos, P < 0.05). The number of Pep44-immunized females that produced embryos was reduced by 64%. Identification and characterization of possum infertility-relevant epitopes on possum ZP2 protein will assist development of safe, humane, and possum-specific immunocontraceptive vaccines for controlling the introduced possums in New Zealand.2. A simplified method for combined immunohistochemistry andin-situ hybridization in fresh-frozen, cryocut mouse brain sections. Sathyanesan Samuel Newton et. alBrain Research Protocols, 9 (3), 214-219 (2002)A method is described to perform combined immunohistochemistry and in situ hybridization in mouse brain sections. The protocol is specific to sections mounted on glass slides. In contrast to earlier methods that require either paraffin embedding or perfusion ofthe brain with paraformaldehyde, this protocol can be carriedout on fresh-frozen, cryostat cut post-fixed sections. This simple and concise protocol increases the applicability of the techniqueas the RNAse-free immunodetection of antigen is useful by itself for immunologically identifying specific cells of interest and then examining gene expression in those cells using techniques such as real-time PCR and microarray analysis. The use of fresh-frozen, cryocut sections enables reliable detection of easily perturbable post-translational modifications such as phosphorylation and improves the quality of results obtained in subsequent in situ hybridization by reducing the background signal and interference from lower cell layers. Inducible transgenic mice that express either a dominant negative mutant form of the cAMP response element binding protein (mCREB) or CREB, in discrete brain regions, were used in this study. The combined immunohistochemistry and in situ hybridization protocol was used to examine colocalization of enkephalin or dynorphin mRNA, both downstream targets of CREB-mediated gene expression, in cells expressing transgenic mCREB or CREB. HistoChoice Tissue FixativeJ Melrose et. alBiotechnic and Histochemistry, 83(1), 47-53 (2008) Histochoice is a proprietary nontoxic, non-cross-linking fixative designedby the manufacturer to replace formaldehyde based fixation protocols. We compared Histochoice and formalin fixation for several cartilaginous tissues including, articular and growth plate cartilage, meniscus and intervertebral disc. The tissues were stained with general histology stains including toluidine blue for tissue proteoglycans, picrosirius red to evaluate collagenous organization, and hematoxylin and eosin to assess cell morphology. The chondroitin sulfate and heparin sulfate substituted proteoglycans aggrecan and perlecan were also immunolocalized in some of the tissues to provide a comparison. Histochoice did not fix deep into the tissue blocks resulting in focal loss of aggrecan and other matrix components from the more central regions of the blocks. This was evident in toluidine blue stained sectionsof immature tibial articular cartilage where loss of glycosaminoglycan was significant in Histochoice fixed tissues. Histochoice fixation worked well, however, in the aggrecan and perlecan immunohistology applications where its non-cross-linking traits were conducive to epitope retrieval and identification by primary antibodies to extracellular matrix components.To place an order or receive technical assistance Order/Customer Service: /orderTechnical Service: /techserviceSafety-related Information: /safetycenter Copyright © 2017 Merck KGaA. All Rights Reserved. Merck and the Vibrant M are trademarks of Merck. HistoChoice is a registered trademark and Sigma-Aldrich is a trademark of Sigma-Aldrich Co. LLC. or its affiliates.Lit. No. sh8707 2017 - 0672309/2017。

– Bimolecular system (Redox System)
• Redox system rxn
• Born and Metal alkyl initiators of free radical polymerization • Photochemical initiator (photoinitiator) • Radiation source
• Classification • Carothers • Addition polymer • Condensation polymer • Flory • Step-growth polymer • Chain-growth polymer
Lecture 3
• • • • •
Gel point Thermosetting Thermoplastic Oligomer Telomer Telechelic polymer • Dendrimer 樹枝狀高分子 • Rotaxanes
2. Free-Radical Chain-Growth Polymerization 2.1 Free-Radical Chain-Growth Polymerization Process
– Kinetic Relationship in Free-Radical Polymerization – Reaction Leading to Formation of Initiating Free-Radicals
• Initiators
– 實驗室通常用3種 Peroxides 過氧化物 Azo compounds Redox Systems
• Thermal decomposition of compounds

Supplementary informationAccess to a main alphaherpesvirus receptor, located basolaterally in the respiratory epithelium, is masked by intercellular junctionsJolien Van Cleemput, Katrien C.K. Poelaert, Kathlyn Laval, Roger Maes, Gisela S. Hussey, Wim Van den Broeck, Hans J. Nauwynck.Supplementary experimental proceduresEHV1 purification and Dio-labellingCulture fluids of EHV1-infected RK-13 cells were clarified by centrifugation at 60,000g for 2h at 4°C. The virus pellet was pooled onto a discontinuous OptiPrep™ gradient (Sigma-Aldrich, St. Louis, MO, USA) containing 10-30% (w/v) of iodixanol and centrifuged at 100,000g for 2.5h at 4°C. After centrifugation, purified opalescent virus bands were harvested at the interface of the 15% and 20% layers. To ensure efficient virus lipophilic labelling, the buffer was exchanged to HNE buffer (5 mM HEPES, 150 mM NaCl, 0.1 mM EDTA, pH 7.4) by the use of a 50K filter device (Millipore corporation, Bedford, MA, USA). While vortexing, 2 nM of 3,3’-Dioctadecyloxacarbocyanine perchlorate (Dio) dissolved in DMSO (Molecular probes, Oregon, USA) was added to the virus. Subsequently, unbound Dio was removed by centrifugation onto a MicroSpin™ G-50 fine column (GE Healthcare, Buckinghamshire, UK). The degree of Dio-labelled virus purity (>90%) was evaluated by simultaneous immunofluorescent staining of EHV1 gB with mouse monoclonal antibody 3F6 (kindly provided by Prof. U. Balasuriya, University of Kentucky, USA) and quantitative analysis by confocal microscopy.Tissue collection and processingRespiratory mucosal explant isolation and cultivationThe respiratory mucosa was stripped from the underlying cartilage and washed in PBS to remove excess blood. Tissues were cut into small square pieces (25 mm2), placed with the epithelial side facing upwards onto fine-meshed gauzes and cultured in a 37°C, 5% CO2, humidified incubator for 24h at air-liquid interface in serum-free medium containing DMEM/RPMI (Invitrogen, Paisley, UK), supplemented with 0.1 mg/mL gentamicin, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.25 µg/mL amphotericin B.EREC isolation and cultivationTracheae were trimmed upon arrival in the lab and washed in PBS to remove excess blood. Tissues were submerged into an enzyme mix of 1.4% pronase (Roche Diagnostics Corporation, Basel, Switzerland) and 0.1% deoxyribonuclease I (Sigma-Aldrich) in calcium- and magnesium-free PBS supplemented with 0.45% glucose (VWR International, Leuven, Belgium), 1% sodium pyruvate (Invitrogen), 100 U/mL penicillin and 0.1 mg/mL streptomycin for 48h at 4°C. Detached cells were then incubated in DMEM/F12 (Invitrogen), containing 1% MEM non-essential amino-acids (Invitrogen), 2.4 µg/mL insulin (Sigma-Aldrich), 100 U/mL penicillin and 0.1 mg/mL streptomycin in a plastic petri dish for 2h to reduce fibroblast contamination by adherence. Isolated EREC were either seeded immediately or stored in liquid nitrogen at a density of 2∙106cells per cryovial until further use. EREC were seeded at a concentration of 1.8∙106 cells/insert overnight into type IV collagen-coated (Sigma-Aldrich) 0.4µm pore size transwell cell culture wells (Costar, Corning, Fisher Scientific, Fair Lawn, USA) in DMEM/F12 (Invitrogen), supplemented with 5% non-heat inactivated FCS (Invitrogen), 1% MEM non-essential amino-acids, 100 U/mL penicillin, 1 mg/mL streptomycin, and 1.25 µg/mL amphotericin B. The next day, seeding medium was removed and the bottom platewells were filled with DMEM/F12, containing 2% Ultroser G (Pall Life Sciences; Pall Corp., Cergy, France), 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 1.25 µg/mL amphotericin B (EREC medium). The transwell, comprising the apical surface of the EREC, was left empty to mimic an air-liquid interface. EREC were incubated in a 37°C, 5% CO2 humidified incubator and medium was changed every 1-2 days until full differentiation. After 5-7 days, the EREC attained a trans-epithelial electrical resistance (TEER) of ~500-700 Ω∙cm-2. TEER was measured using an epithelial voltohmmeter (Millipore). The net resistance was calculated by subtracting the background resistance and multiplying the resistance by the surface area of the membrane.Disruption of intercellular bridges of respiratory mucosal explantsFirst, 24-well culture dishes were filled with 1 mL of a solution containing 50% sterile 3% agarose (low temperature gelling; Sigma-Aldrich) and 50% 2X MEM (Invitrogen). Explants were placed onto the solidified agarose with the epithelial surface facing upwards. Additional agarose was added until the lateral surfaces of the mucosa were fully occluded. Explants were then exposed for 1h at 37°C to different drugs (8 mM EGTA, 500 mM NAC, 20 mM DTT or 50 mM β-mercaptoethanol in PBS). PBS supplemented with calcium and magnesium was used as a control. Finally, explants were washed 3 times to remove excess drugs and were fixed in phosphate-buffered 3.5% formaldehyde solution, either immediately or after an additional 24h incubation. An automated system was used for paraffin embedding of the samples (Thermo Scientific™ STP 120 Spin Tissue Processor). Eight µm paraffin sections were first deparaffinised in xylene, then rehydrated in descending grades of alcohol, subsequently stained with haematoxylin-eosin, dehydrated in ascending grades of alcohol and xylene and finally mounted with DPX (Sigma-Aldrich). Ten pictures on five different sections per treated explant were taken with an Olympus IX50 light microscope fitted with 40X objective. The percentage of intercellular space in the epithelium was measured using ImageJ software (ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA). The region of interest (ROI, i.e. the epithelium) was drawn manually for each picture in the “ROI manager tool”. Next, the threshold value to distinguish blank spaces from cellular material was determined and the percentage of blank spaces between the cells (i.e. the intercellular space) was calculated. Immunofluorescent staining and confocal microscopyRespiratory mucosal explantsSixteen µm thick cryosections were cut using a cryostat at -20°C and loaded onto 3-aminopropyltriethoxysilane-coated (Sigma-Aldrich) glass slides. Slides were then fixed in 4% paraformaldehyde for 15min and subsequently permeabilized in 0.1% Triton-X 100 diluted inPBS. Non-specific binding sites were blocked by 15min incubation with avidin and biotin (Invitrogen) at 37°C. To label late viral glycoproteins, a polyclonal biotinylated horse anti-EHV1 was used for 1h at 37°C1, followed by incubation with streptavidin-FITC® (Invitrogen) for 1h at 37°C. The basement membrane of the tissues was stained with monoclonal mouse anti-collagen VII antibodies (Sigma-Aldrich), followed by secondary Texas Red® labelled goat anti-mouse antibodies (Invitrogen). Nuclei were detected by staining with Hoechst 33342 (Invitrogen). Slides were mounted with glycerol-DABCO and analysed using a Leica (TCS SPE) confocal microscope. The total number of plaques was counted on 50 cryosections and plaque latitude was measured using the Leica confocal software package. Five cryosections per explant were completely photographed and the percentage of infection in the epithelium (i.e. ROI) was determined using Image J software. The ROI (i.e. the epithelium) was drawn manually for each picture in the “ROI manager tool”. Next, the threshold value to distinguish the FITC positive signal from the background signal was determined and the percentage of FITC positive signal (i.e. infection) was calculated.Cellular glycosaminoglycans, heparan sulfate, chondroitin sulfate A and B and sialic acids were respectively stained with a monoclonal mouse anti-heparan sulfate antibody (10E4; Ambsio, Abingdon, UK), a monoclonal mouse anti chondroitin sulfate (CS-56; Bio-rad; Oxford; UK) or biotinylated Maackia Amurensis lectin (Vector Laboratories; Peterborough; UK) followed by a goat anti-mouse FITC® antibody or streptavidin-FITC®.ERECAntibodies were incubated directly in the transwells for 1h at 37°C. Cells were first incubated with a 1:1,000 dilution of a polyclonal rabbit anti-IEP antibody, kindly provided by Dr. D. O’Callaghan, Louisiana State University, USA. The diluent used was PBS containing 10% negative goat serum. This was followed by incubation with a goat anti-rabbit IgG FITC®conjugated antibody (Invitrogen). Nuclei were counterstained with Hoechst 33342 for 10minat 37°C. Transwell membranes were excised from the culture inserts and mounted on glass slides using glycerol-DABCO. Slides were examined using a Leica confocal microscope. The total number of plaques was counted on 5 random fields of approximately 3∙104 cells per insert. Plaque latitude was measured on 10 individual plaques using the Leica confocal software package.Enzymatic removal of cell surface N-linked glycans and sialic acids prior to EHV1 inoculationPNGase F (New England Biolabs, Ipswich, UK) removes complex, hybrid and oligomannose N-glycosylations and was applied onto apical or basolateral EREC surfaces for 12h at a concentration of 25,000 U/mL, diluted in EREC medium, supplemented with 10% glycobuffer (New England Biolabs; Ipswich; UK). Neuraminidase from Vibrio cholera (Sigma-Aldrich) has a broad substrate spectrum for sialic acids and was used for 1h at 50 mU/mL in PBS. The influenza A/Equine/Kentucky/98 (H3N8) strain served as a positive control during neuraminidase treatment of EREC. The virus was propagated in embryonated eggs and titrated onto Madin-Darby canine kidney (MDCK) cells. Influenza A strains can easily infect MDCK cells through interaction with cell-associated sialic acids, which can be removed with neuraminidase2. Equine influenza virus (EIV) A nucleoprotein was stained with the mouse monoclonal antibody HB-65 (ATCC) and subsequently visualized with FITC®-labelled secondary goat anti-mouse IgG antibodies. Nuclei were counterstained with Hoechst 33342 and coverslips were mounted using glycerol-DABCO. The percentage of positive cells was calculated based on the total number of positive cells out of 300 randomly selected cells. Correct cleavage of the respective sialic acids was corroborated by immunofluorescent staining of EREC with biotinylated Maackia Amurensis Lectin II (Vector laboratories). The complex was subsequently stained with Streptavidin-FITC® (Invitrogen). Ten z-stack confocal pictures were taken to distinguish apical from basolateral treatment. The means of the fluorescent apicalor basolateral signals were compared with Image J software and dropped significantly after treatment with neuraminidase, compared to control. Following enzymatic treatment, cells were washed 3 times with DMEM/F12 and the inoculum was delivered on top of the respective surfaces for 1h at 37°C. Concurrently, and as a positive control for enzymatic treatment, MDCK cells were treated with neuraminidase before inoculation with EIV. After 1h inoculation, unbound virus particles were removed by washing and cells were incubated for 10 hours before fixation in methanol, as described above.Supplementary figuresFigure S1. Cell viability in respiratory mucosal explantsTUNEL-staining data of tracheal ME (left) and nasal ME (right) after different treatments. Three independent experiments were performed and data are represented as means + SD. The lower case letters indicate significant (P<0.05) differences in the epithelium, while the upper case letters indicate significant differences in the lamina propria.Figure S2. The disruption of intercellular bridges in EREC(a) Trans-epithelial electrical resistance of EREC prior to treatment and after 30min treatment with PBS (control) or EGTA. Three independent experiments were performed and the data are represented as means + SD. Different letters indicate significant (P<0.05) differences. (b) EMA-staining confirmed no significant (P<0.05) differences in cell viability after different treatments, when compared to cell viability prior to treatment. Three independent experiments were performed and data are represented as means + SD. Different letters indicate significant (P<0.05) differences.Figure S3. Validation neuraminidase assay(a) During enzymatic treatment, MDCK cells were similarly pre-treated with neuraminidase before inoculation with EIV. Cells were fixed at 6hpi in methanol and EIV-positive cells were visualized using the monoclonal HB65 antibody. Five random fields of approximately 100 cells were screened to calculate the percentage of EIV-positive cells. Experiments were performed in triplicate. Data are represented as means + SD and different letters represent significant differences. (b) Correct sialic acid-cleavage from EREC surfaces was confirmed by confocal analysis of 10 different z-stacks per treatment. EREC were grown to confluency on transwells and treated at either the apical surface (left) or basolateral surface (right) with neuraminidase or control PBS for 1h at 37°C. Cells were then fixed in PFA and permeabilized in Triton X, sialic acids were stained with biotinylated Maackia Amurensis lectin and the percentage of fluorescent signal in either the apical or the basolateral domain of EREC was calculated. Data are represented as mean + SD. Significant differences after apical exposure are indicated by different lower case letters and after basolateral exposure by different upper case letters.Supplementary references1 van der Meulen, K., Vercauteren, G., Nauwynck, H. & Pensaert, M. A local epidemicof equine herpesvirus 1-induced neurological disorders in Belgium. Vlaams Diergeneeskundig Tijdschrift72, 366-372 (2003).2 Stray, S. J., Cummings, R. D. & Air, G. M. Influenza virus infection of desialylatedcells. Glycobiology10, 649-658 (2000).。