1. Preparation of Slides

 A. Cell Lines

• Grow cultured cells on sterile glass cover slips or slides overnight at 37 º C
• Wash briefly with PBS
• Fix as desired. Possible procedures include:10 minutes with 10% formalin in PBS (keep wet)5 minutes with ice cold methanol, allow to air dry5 minutes with ice cold acetone, allow to air dry
• Wash in PBS

B. Frozen Sections

• Snap frozen fresh tissues in liquid nitrogen or isopentane pre-cooled in liquid nitrogen, embedded in OCT compound in cryomolds. Store frozen blocks at - 80 ºC.
• Cut 4-8 um thick cryostat sections and mount on superfrost plus slides or gelatin coated slides. Store slides at - 80 ºC until needed.
• Before staining, warm slides at room temperature for 30 minutes and fix in ice cold acetone for 5 minutes. Air dry for 30 minutes.
• Wash in PBS

C. Paraffin Sections

• Deparaffinize sections in xylene, 2x5min.
• Hydrate with 100% ethanol, 2x3min.
• Hydrate with 95% ethanol, 1min.
• Rinse in distilled water.
• Follow procedure for pretreatment as required.

2. Pretreatments of Tissue Sections

Antigenic determinants masked by formalin-fixation and paraffin-embedding often may be exposed by epitope umasking, enzymatic digestion or saponin, etc. Do not use this pretreatment with frozen sections or cultured cells that are not paraffin-embedded.

3. Procedure

Note: prior to perform double labeling, it is important to test each primary antibody individually and select the best pretreatment(s) for each antibody. It will be ideal if the two primary antibodies require same pretreatment. Otherwise, one should do a further test by treating sections with both pretreatments and then immunostain for each antibody individually. If both antibodies survive the “double pretreatments”, you are ready for immunohistochemistry double staining. Another alternative is to do pretreatments separately for each antibody staining.

1. Rinse Sections in PBS-Tween 20 for 2x2 min.
2. 1^st Serum Blocking: incubate sections in normal serum – species same as secondary antibody (for example: 1^st primary antibody is mouse and 1^st secondary antibody is horse anti-mouse, so horse normal serum blockshould be used).
3. 1^st Primary Antibody: incubate sections in 1^st primary antibody (mouse) at appropriate dilution in antibody diluent for 1 hour at room temperature. Notes: (1) do not rinse between step 2 and 3; (2) room temperature means 22-25 °C. If too low, longer incubation time is needed; (3) some antibodies may require overnight incubation.
4. Rinse in PBS-Tween 20 for 3x2 min.
5. 1^st Secondary Antibody: incubate sections in FITC conjugated horse anti-mouse secondary antibody in PBS for 20-30 minutes at room temperature.  
6. Rinse in PBS-Tween 20 for 3x2 min.
7. 2^nd Serum Blocking: use normal mouse serum to block free binding sites of the anti-mouse immunoglobulin.
8. 2^nd Primary Antibody: incubate sections in 2^nd primary antibody (mouse) at appropriate dilution in antibody diluent for 1 hour at room temperature. �
9. Rinse in PBS-Tween 20 for 3x2 min.  
10. 2^nd Secondary Antibody: incubate sections in Texas Red conjugated anti-mouse secondary antibody in PBS for 20-30 minutes at room temperature.  
11. Rinse in PBS-Tween 20 for 3x2 min. 
12. Counterstain with DAPI if desired for 20 minutes at room temperature.
13. Rinse in PBS-Tween 20 for 3x2 min.
14. Coverslip with anti-fade mounting medium.
15. Store slides in dark at 4 °C.

 4. Results

• 1^st Primary Antibody Staining Sites ------------------- green
• 2^nd Primary Antibody Staining Sites------------------- red  
• Double Staining Sites ----------------------------------- yellow
• Counterstained Nuclei ---------------------------------- light blue