FITC免疫荧光检测试剂盒小鼠FITCImmunofluorescence

小鼠c-fos试剂盒使用方法检测范围：96T20pg/ml-480pg/ml使用目的：本试剂盒用于测定小鼠血清、血浆及相关液体样本中c-fos含量。

实验原理本试剂盒应用双抗体夹心法测定标本中小鼠c-fos水平。

用纯化的小鼠c-fos抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依次加入c-fos，再与HRP标记的c-fos抗体结合，形成抗体-抗原-酶标抗体复合物，经过彻底洗涤后加底物TMB显色。

TMB在HRP酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。

颜色的深浅和样品中的c-fos呈正相关。

用酶标仪在450nm波长下测定吸光度（OD值），通过标准曲线计算样品中小鼠c-fos 浓度。

试剂盒组成标本要求1．标本采集后尽早进行提取，提取按相关文献进行，提取后应尽快进行实验。

若不能马上进行试验，可将标本放于-20℃保存，但应避免反复冻融2．不能检测含NaN3的样品，因NaN3抑制辣根过氧化物酶的（HRP）活性。

操作步骤1.标准品的稀释：本试剂盒提供原倍标准品一支，用户可按照下列图表在小试管中进行稀2.加样：分别设空白孔（空白对照孔不加样品及酶标试剂，其余各步操作相同）、标准孔、待测样品孔。

在酶标包被板上标准品准确加样50μl，待测样品孔中先加样品稀释液40μl，然后再加待测样品10μl（样品最终稀释度为5倍）。

加样将样品加于酶标板孔底部，尽量不触及孔壁，轻轻晃动混匀。

3.温育：用封板膜封板后置37℃温育30分钟。

4.配液：将30倍浓缩洗涤液用蒸馏水30倍稀释后备用5.洗涤：小心揭掉封板膜，弃去液体，甩干，每孔加满洗涤液，静置30秒后弃去，如此重复5次，拍干。

6.加酶：每孔加入酶标试剂50μl，空白孔除外。

7.温育：操作同3。

8.洗涤：操作同5。

9.显色：每孔先加入显色剂A50μl，再加入显色剂B50μl，轻轻震荡混匀，37℃避光显色15分钟.10.终止：每孔加终止液50μl，终止反应（此时蓝色立转黄色）。

fitc的激发波长和发射波长
FITC指Fluorescein isothiocyanate，是一种有机化学物质，它具有荧光特性，可以用微量添加到物质中，从而使其具有调节特性。

FITC的激发波长是495 nm，发射波长是520 nm。

FITC的主要用途有：
一、用于染色细胞。

FITC可以充当FLUOR和CFAs的凝胶电泳染色剂，有助于更好地解释实验结果，而不会影响实验的灵敏度。

另外，FITC还可以通过免疫细胞染色法来识别特定的细胞，使其与研究相关的特定信号互相关联。

二、用于组织及细胞构建。

FITC可以把特定的核酸和蛋白质附着在特定的细胞表面，以便标记指定的细胞，例如脂肪细胞、神经元和血管细胞等。

三、用于真核生物中的遗传测定。

FITC可以运用在荧光原位杂交技术（FISH）中，将特定的核酸链结合在特定的序列上，以帮助实验者测定基因的表达和遗传变异情况。

四、用于EMT鉴定。

FITC可以配合其他技术，如荧光技术、传递电子显微镜和免疫细胞化学分析，用于鉴定多种疾病的机制，例如肝癌、肠癌和胰腺癌等。

FITC有着许多广泛的用途，激发波长495nm，发射波长为520nm，并且可以将特定的核酸和蛋白质附着在特定的细胞表面，以帮助实验者做出合理的判断，获得准确的实验结果。

免疫荧光法检测干细胞标志物英文回答：Immunofluorescence is a widely used technique to detect and visualize specific proteins or molecules in biological samples. It utilizes the specific binding of antibodies to their target molecules, followed by the detection of these antibodies using fluorescent markers.In the context of stem cell research, immunofluorescence can be used to detect and identify specific markers that are characteristic of stem cells. Stem cells are undifferentiated cells with the ability to differentiate into various cell types. They express certain proteins or molecules that are unique to stem cells, and these markers can be detected using immunofluorescence.For example, one commonly used stem cell marker is Oct-4. Oct-4 is a transcription factor that plays a crucialrole in maintaining pluripotency in embryonic stem cells.To detect Oct-4 using immunofluorescence, researchers would first incubate the stem cells with a primary antibody that specifically binds to Oct-4. This primary antibody is typically derived from a different species than the sample (e.g., mouse anti-Oct-4 antibody for human stem cells). After washing away any unbound antibodies, a secondary antibody conjugated to a fluorescent dye (e.g., FITC or Alexa Fluor) is added. This secondary antibody recognizes and binds to the primary antibody, allowing for the visualization of Oct-4 under a fluorescence microscope. The presence of Oct-4 in the stem cells can be identified by the green fluorescence emitted by the FITC or Alexa Fluor dye.In addition to Oct-4, there are many other markers that can be detected using immunofluorescence to identify and characterize stem cells. These include Nanog, Sox2, SSEA-4, and CD133, among others. Each of these markers has its own specific antibody that can be used in immunofluorescence experiments.Immunofluorescence is a powerful tool in stem cellresearch as it allows for the visualization and identification of stem cells based on their unique markers. It provides valuable information about the presence, location, and abundance of stem cells in a sample. This information is crucial for understanding stem cell biology and for the development of stem cell-based therapies.中文回答：免疫荧光法是一种广泛应用于生物样本中特定蛋白质或分子检测和可视化的技术。

FluoroTag™FITC Conjugation KitProduct Number FITC1Storage Temperature 2−8 °CTECHNICAL BULLETINProduct DescriptionThe FluoroTag FITC Conjugation Kit is suitable for the conjugation of polyclonal and monoclonal antibodies with fluorescein isothiocyanate (FITC) for use in immunohistochemistry and immunofluorescence studies utilizing flow cytometry. It may also be used for conjugation of FITC to peptide hormones, cytokines, growth factors, and other proteins. The kit contains sufficient reagents for at least 5 conjugations. Fluorescein isothiocyanate (FITC), Isomer I is among the most widely used fluorescent labeling reagents due to the fluorophore’s high quantum efficiency and conjugate stability. FITC has an absorption maximum at 495 nm and emission maximum at 525 nm. FITC reacts with free amino groups of proteins to form stable conjugates (see Figure 1). FITC-protein conjugates, in particular FITC labeled antibodies, are used as specific probes in immunocytochemistry and flow cytometry applications.1,2Biologically active FITC-conjugates of peptide hormones and growth factors have been successfully prepared which identify receptors on target cells.3,4FITC has also been used as a site-specific probe for several other proteins.5,6,7The use of optimal labeling conditions is recommended. Overlabeling of proteins generally results in altered specificity, aggregation and/or precipitation of the protein. Fluorescent labeling of antibodies with high fluorophore to antibody ratios (molar F/P >6) usually results in increased non-specific binding (fluorescent background) and decreased quantum yield due to the fluorophore self-quenching effect.The FluoroTag Kit includes detailed procedures for both small and large scale conjugation of FITC to antibody. Small scale FITC conjugations are performed using three different molar ratios of FITC to antibody. Based on the molar ratio that gives the most satisfactory result, the large scale procedure can then be performed to optimally label the protein. The labeled protein is purified from the unconjugated fluorescein by a quick Sephadex®G-25M column. The F/P molar ratio of the purified protein is then determined by measuring the absorbance at 280 nm and at 495 nm.Reagents and Materials Provided• Fluorescein isothiocyanate, Isomer I (FITC), F7250.Five amber vials each containing 2.0 mg oflyophilized fluorescein isothiocyanate.• 0.1M Sodium Carbonate-Bicarbonate Buffer, pH9.0, C0688. Five capsules containing powder.• Phosphate Buffered Saline (PBS), P3813. Five packages containing powder.• Gel filtration columns. Two columns prepacked with Sephadex G-25M. These serve to separateunreacted FITC from the conjugate and for bufferexchange. The columns are preswollen in watercontaining 0.15% Kathon®CG/ICP II aspreservative. The gel filtration columns are supplied for two different reaction scales:Small Scale (Column A), B7533:One columnprepacked with Sephadex G-25M. The bed volume of the column is 3.5 ml and the bed height is2.6cm. The maximal sample volume is 0.3 ml.Large Scale (Column B), B4783:One columnprepacked with Sephadex G-25M. The bed volume of the column is 9.1 ml and the bed height is 5cm.The maximal sample volume is 1.5 ml.Reagents and Equipment Required but Not Provided• Standard glass vials (1.5−2 ml capacity) equipped with stirring bars.• Two dilution vials are needed for each conjugation.• Vortex mixer.• Standard glass tubes (12 X75 mm or 13 X100 mm) to collect fractions from Sephadex G-25M columns.• Aluminum foil -To protect reaction and FITC labeled protein from intense light.• Quartz cuvette -1 cm path length• UV/Visible spectrophotometer.2Precautions and DisclaimerThis product is for R&D use only, not for drug, household, or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.Preparation Instructions0.1 M Sodium Carbonate-Bicarbonate Buffer, pH 9.0,C0688-Add contents of one capsule to 50 ml of deionized water to make 0.1M sodium carbonate-bicarbonate buffer, pH9.0. This is used to dissolve FITC and to buffer the conjugation reaction. Phosphate Buffered Saline (PBS), P3813-Mix contents of one package with 800 ml of deionized water. Adjust volume to 1 liter to make 10 mM sodium phosphate buffer, 27 mM KCl, 138 mM NaCl, pH 7.4. This serves as an equilibration buffer for the Sephadex G-25 columns, for the elution of the labeled protein from the column, and for the final dilution of the labeled protein.Storage/StabilityStore at 2−8 °C.ProcedureThe protocol outlined describes the labeling of 1 mg of IgG at 5 mg/ml with FITC (Small Scale Conjugation Procedure). The procedure can be scaled up to 5 mg of IgG maintaining the same concentration and molar ratio of the reagents (Large Scale Conjugation Procedure). It is important to consider that the number and surface availability of amine groups (primarily -amine groups of lysine residues) vary greatly among proteins and even among different IgGs. This may result in a large variability of the level of labeling. Testing different FITC to antibody molar ratios to determine the optimal levels of labeling of the antibody is recommended.Small Scale Conjugation Procedure(1.0 mg IgG)This procedure describes the conjugation of FITC to1mg of IgG, using one of the following molar ratios in the reaction mixture: 5:1, 10:1 and 20:1 of FITC(MW389) to IgG (MW 150,000). The labeling is performed in a final reaction volume of 0.25 ml. In general, these reaction molar ratios result in fluorescein-antibody conjugates with F/P ratios of 1−2, 2−4 and 3−6, respectively. This procedure can be modified if a protein of a different molecular weight (or a different amount of IgG) is used in the labeling reaction. (See Table 2 reaction mixture ratios for whole IgG, IgM, or antibody fragments.)1.Dissolve the contents of one sodium carbonate-bicarbonate capsule (C0688) in 50 ml of deionized water. The pH of this buffer should be 9.0 ±0.1(See Application Notes 1&2).2.Prepare at least 0.25 ml of antibody solution at5.0mg/ml in 0.1 M carbonate-bicarbonate buffer,pH 9.0 (See Application Notes 3, 4 & 5). The A280of an IgG solution at 1.0 mg/ml is 1.4 (1.0 cm path length).3.Add 0.2 ml (1.0 mg) of the antibody solution intoeach reaction vial labeled “5:1”, “10:1”, or “20:1”. 4.Reconstitute one vial of FITC (F7250) in 2 ml of0.1M carbonate-bicarbonate buffer and vortex untilall FITC has dissolved. Label vial “20:1 FITC”. The solution should be freshly prepared before eachconjugation and used within 5 minutes (SeeApplication Note 6). The "20:1 FITC" solution isfurther used to prepare the "5:1" and "10:1"solutions.5.Prepare the required dilution of FITC in 0.1Mcarbonate-bicarbonate buffer as directed inTable1.Table 1.Dilution of FITC in 0.1M Carbonate-Bicarbonate Buffer6.Add 50 µl of the appropriate FITC dilution dropwisewhile stirring to the correspondingly labeledreaction vial.pletely cover the reaction vial with aluminumfoil to protect from light.8.Incubate all reaction vials for 2 hours at roomtemperature with gentle stirring.Isolation of labeled protein1.Empty contents of PBS package (P3813) into asuitable container. Add 800 ml of distilled ordeionized water and mix. Adjust to final volume of 1,000 ml.bel Sephadex G-25M, column A (B7533), “5:1”,“10:1”, or “20:1”. Support column over a suitable(100ml) beaker.3.Remove cap from the top of the column, cut openlower tip of column and let excess of liquid flowthrough. The column will not run dry.34.Equilibrate the column with 12 ml of PBS solution(6 X2 ml). If the column is not immediately used,close with top and bottom caps and store at 2−8 °C.5.Apply reaction mixture to top of column gel bed andcollect the flow through (Fraction 1)6.Elute the column with 2.5 ml of PBS, collecting0.25ml fractions (10 X0.25 ml). Monitor theabsorbance of each fraction at 280 nm. Two bands will be visible during elution. The conjugate ispresent in the first band (fractions 6−8)(See Application Note 7).7.Pool the main fractions. Do not collect fractions withA280<0.2.8.Wash the column with 35 ml (10 X column volumes)of PBS to remove unbound fluorophore. This issufficient to regenerate the column.9.For prolonged storage, wash the column with 10mlof PBS containing 0.05% sodium azide and storecapped at 2−8 °C, with 1 ml buffer above the gel. 10.Determine the fluorescein/protein ratio (F/P) of theconjugate using a spectrophotometer as described in Determination of Fluorescein/Protein Molar Ratio (F/P).11.For storage of the conjugate after determination ofthe F/P molar ratio, add 1% (w/v) BSA and 0.1%(w/v) sodium azide to the conjugate. Store at2−8°C, protected from light.Large Scale Conjugation Procedure(5.0 mg IgG)This procedure describes the scale-up of the conjugation reaction of FITC to 5 mg of IgG, using the molar ratio, which gives the most satisfactory results as obtained from the “Small Scale Conjugation Procedure.” The labeling is performed maintaining the same concentrations and ratios of reagents used previously. The reaction is performed in a final reaction volume of 1.25 ml.1.Add 1.0 ml (5.0 mg) of the antibody solution to areaction vial labeled “5:1”, “10:1”, or “20:1”.2.Reconstitute one vial of FITC (F7250) in 2 ml of0.1 M carbonate-bicarbonate buffer and vortex untilall FITC has dissolved.3.Prepare 10:1 or 5:1 dilution of FITC in 0.1Mcarbonate-bicarbonate buffer as directed inTable1, if necessary.4.Add 250 µl of the appropriate FITC dilutiondropwise while stirring to the reaction vial.pletely cover the reaction vial with aluminumfoil to protect from light.6.Incubate reaction vial for 2hours at roomtemperature with gentle stirring.Isolation of labeled proteinbel Sephadex G-25M, column B (B4783), “5:1”,“10:1”, or “20:1”. Support the column over asuitable (100 ml) beaker.2.Remove cap from the top of the column, cut openlower tip of column and let excess of liquid flowthrough. The column will not run dry.3.Equilibrate the column with 30 ml of PBS solution(6 X5 ml). If the column is not immediately used,close with top and bottom caps and store at 2−8 °C.4.Apply reaction mixture to top of the column gel bedand collect flow through (Fraction 1).5.Elute column with 10 ml of PBS, collecting 1.0 mlfractions (10 X1 ml). Monitor the absorbance ofeach fraction at 280 nm. Two bands will be visible during elution. The conjugate is present in the first band (fractions 3−5). (See Application Note 7.)6.Pool the main fractions. Do not collect fractionswith A280<0.4.7.Wash the column with 50 ml of PBS solution toremove unbound fluorophore. This is sufficient toregenerate the column.8.Preserve the column as instructed in step 9 of thesmall scale “Isolation of Labeled Protein” section. Determination of Fluorescein/Protein Molar Ratio (F/P) The F/P molar ratio is defined as the ratio of moles of FITC to moles of protein in the conjugate.To determine this ratio, it is necessary to first determine the absorbance of the conjugate sample at 280 nm and then at 495 nm.1.Place the conjugate sample in a quartz cuvette.For the large scale conjugation, dilute 0.1 ml of the FITC conjugate in 0.9 ml of PBS containing sodium azide. Read the absorbance of the conjugatesample at 280 nm and 495 nm. The absorbancereading of the conjugate sample should be between0.2 and 1.4 at 280 nm. If the absorbance reading isoutside this range, adjust the sample dilutionaccordingly.2.From the absorbance readings (A280and A495) ofthe conjugate sample, calculate the F/P of thefluorescein-IgG conjugate according to the84The protein concentration of the fluorescein-IgGFITC, use the general formula below, substituting the appropriate values for the particular protein:MW is the molecular weight of the protein.389 is the molecular weight of FITC.195 is the absorption E 0.1% of bound FITC at 490 nm at pH 13.0.(0.35 x A 495) is the correction factor due to the absorbance of 280(For E 0.1%and C values, see Table 2)FITC at 280 nm.8E 0.1%is the absorption at 280 nm of a protein at 1.0mg/ml.280280Application Notes1.Do not store sodium carbonate-bicarbonate buffermore than 1 week at 2−8 °C. The pH of the buffer may change upon storage. It is advised that fresh buffer be made just before use.2.The conjugation of FITC to proteins is affected bythe reaction conditions (concentration, temperature, pH).3.When conjugating antibodies with FITC, the startingmaterial should be free of contaminating serum proteins. Affinity isolated antibodies, IgG fractions, or Protein A purified immunoglobulin are generally acceptable.4.Protein solutions should not be prepared in bufferscontaining amines such as Tris, glycine or sodium azide since they inhibit the labeling reaction. If the buffer contains amines or sodium azide, dialyze protein solution (1 ml) against PBS, pH 7.4(1,000ml), overnight at 2−8 °C. Avoid dialysis at high pH values (>8.0−8.5) as this may be harmful to some proteins.5.If the antibody is in PBS pH 7.4 (without azide), add1 M carbonate-bicarbonate buffer (1 capsule (C0688) in 5 ml deionized water) to a final concentration of 0.1 M (e.g., 0.1 ml of 1 Mcarbonate-bicarbonate buffer to 0.9 ml IgG solution at 5.0 mg/ml).6.FITC is not stable in aqueous solutions. Do notstore FITC stock solutions.7.The second band containing unbound fluorophoreis retained on the column and will elute only with subsequent PBS washes.References1.Staines, W., et al., Three-colorimmunofluorescence histochemistry allowing triple labeling within a single section. J. Histochem. Cytochem., 36, 145 (1988).2.Carter, N., in Flow Cytometry: A PracticalApproach, M.G. Ormerod, Ed., IRL Press (1990).3.Carraway, K., et al., Location of the epidermalgrowth factor binding site on the EGF receptor. A resonance energy transfer study. Biochemistry , 29, 8741 (1990).4.Schwartz, J., and Vale, W., Fluorescent andcytotoxic analogs of corticotropin-releasing factor: probes for studying target cells in heterogeneous populations. Methods in Enzymol ., 168, 29 (1989).5.Adams, S., et al., Fluorescence ratio imaging ofcyclic AMP in single cells. Nature , 349, 694 (1991).6.Abbott, A., et al., Immunochemical andspectroscopic characterization of two fluorescein 5'-isothiocyanate labeling sites on Na+, K(+)-ATPase. Biochemistry , 30, 1692 (1991).7.Pavalko, F., and Burridge, K., Disruption of theactin cytoskeleton after microinjection of proteolytic fragments of alpha-actinin. J. Cell Biol.,114, 481 (1991).8.The, T., and Feltkamp, T., Conjugation offluorescein isothiocyanate to antibodies. I. Experiments on the conditions of conjugation. Immunol.,18, 865 (1970).Sephadex is a registered trademark of Pharmacia, Inc. Kathon is a registered trademark of the Rohm & Haas Company.LPG,KTA 12/05-15 Table 2.Reaction Mixture Ratios for Whole IgG, IgM or Antibody Fragments** This corresponds to volume in microliters of the appropriate FITC concentration (5:1, 10:1, or 20:1).***For IgM, a lower molar ratio is recommended as high levels of FITC may result in overlabeling of the antibody and self-quenching of the fluorophore.****For F(ab′) 2 use a 2-fold more concentrated FITC solution than used for IgG.*****For Fab′and Fc use a 4-fold more concentrated FITC solution than used for IgG.6Figure 1.The FITC Labeling ReactionFITC conjugation occurs through the free amino groups of proteins or peptides, forming a stable thiourea bond.Sigma brand products are sold through Sigma-Aldrich, Inc.Sigma-Aldrich, Inc. warrants that its products conform to the information contained in this and other Sigma-Aldrich publications.Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply.Please see reverse side of the invoice or packing slip.。