Immunocytochemistry.doc

中山大学负附属第一医院 肾病实验室Immunocytochemistry Methods, Techniques and ProtocolsPreparation of Coated Slides 1.1 Position clean glass slides in a staining rack1.2 Immerse the slides for 30 min in a large staining dish containing a 1:10 dilution of 0.1% poly-L-lysine solution in deionized water.1.3 Remove the slides and oven dry for 1 hr at 60 C.Cell Culture Slide Preparation4.1 Transfer 200 ul of cell culture to the wells of a chamber slide or slides of your choice (Choice of slide design is often dictated by the experiment. Some slides have four wells, some have eight, some are glass, and some are plastic. Glass is recommended simply because the slide becomes more versatile, acetone can be used as a fixative, and finished slides can be dehydrated in ethanol and cleared in xylene)4.2 Allow cells to grow to confluence with the addition of fresh media.4.3 Wash the slides with PBS 2x5min4.4 Fix cells with any of the below mentioned fixation methods. (see: Cell Fixation as follow )4.5 Rinse 5x2 min in PBS.4.6 Incubate the slides with 0.25-0.5% Triton X-100 in PBS for 10 minutes to permeabilize the membranes (Note: there is no need for a permeabilization step following acetone or methanol fixation).4.7 Rinse 3x5 min in PBS.4.8 Blocking endogenous peroxidase by incubating in 3% H2O2 in PBS for 10-30 minutes (Do this step only if a peroxidase marker is to be used. Omit this step if a fluorescent or AP marker is to be used. 4.9 Rinse in 3x5min in PBS.4.10 Blocking with 2-5% normal serum in5%BSA in PBS for 1 hour RT (Normal serum should be the same species as the secondary antibody is raised).4.11 Incubate with primary antibody diluted in blocking buffer and proceed to routine immunocytochemistry procedure.1. Antibody controls (2nd Ab ONLY )2. Secondary antibody-fluorochrome-labeled in blocking buffer RT 1hr3. wash in PBS 3x5 min4. Incubate in 0.1-1 mg/ml Hoechst(1:10000 )or DAPI(1:400)in PBS (DNA stain) for 5 min. 5. Rinse with PBS. 6. Aqueous mounting medium Cell Fixations1. Acetone Fixation Fix cells in -20C acetone for 5-10 minutes. No permeabilization step needed following acetone fixation.2. Methanol Fixation Fix cells in -20C methanol for 5-10 minutes. No permeabilization step needed following methanol fixation.3. Ethanol Fixation Fix cells in cooled 95% ethanol, 5% glacial acetic acid for 5-10 minutes.4. Methanol-Acetone Fixation Fix in cooled methanol, 10 minutes at 20 C. Remove excess methanol. Permeabilize with cooled acetone for 1 minute at 20 C.5. Methanol-Acetone Mix Fixation 1:1 methanol and acetone mixture. Make the mixture fresh and fix cells at -20 C for 5-10 minutes. 6. Methanol-Ethanol Mix Fixation 1:1 methanol and ethanol mixture. Make the mixture fresh and fix cells at -20 C for 5-10 minutes.7. Formalin Fixation Fix cells in 10% neutral buffered formalin for 5-10 minutes. Rinse briefly with PBS. Permeabilize with 0.5% Triton X-100 for 10 minutes.8. Paraformaldehyde-Triton Fixationo Fix in 3-4% paraformaldehyde for 10-20 minutes. 3-4% Paraformaldehyde (Product Code P 6148 from Sigma) in PBS. Dissolve in PBS, using 5N NaOH and mild heating o FA stock solution: Dissolve 16 g paraformaldehyde in about 80 ml dH2O by stirring at 70 oC (in fume cupboard). Add a few drops of 1 N NaOH to depolymerize the paraformaldeyde. Adjust the pH to about 7.0 and check with pH paper). Cool down to room temperature and bring up to 100 ml. Filter through an 0.45 m Millipore filter and mix with an equal amount of double strength buffer. Divide into convenient aliquots and store frozen at -20oC. Discard after thawing. Rinse briefly with PBS. Permeabilize with 0.5% Triton X-100 for 10 minutes.9. Paraformaldehyde-Methanol Fixation