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 for ImmunoEnzyme Double Staining

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 dilution buffer 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.   Peroxidase Blocking: incubate sections in peroxidase blocking solution for 10 minutes at room temperature.

6.   1^st Secondary Antibody: incubate sections in 1^st biotinylated secondary antibody – horse anti-mouse in PBS for 20-30 minutes at room temperature.

7.   Rinse in PBS-Tween 20 for 3x2 min.

8.   1^st Detection: incubate sections in Streptavidin-HRP in PBSfor 20-30 minutes at room temperature. Note: don’t use serum (including BSA) solution to diluent HRP-Streptavidin, since serum may contain biotin therefore competing Streptavidin binding with biotinylated secondary antibody, therefore reducing binding activity.

9.   Rinse in PBS-Tween 20 for 3x2 min.

10. 1^st Chromogen/Substrate: incubate sections in DAB-blue for 1-3 minutes to produce Dark Blue reaction product.

11. Rinse in PBS-Tween 20 for 3x2 min.

12. 2^nd Serum Blocking: incubate sections in normal serum – species same as secondary antibody (for example: 2^nd primary antibody is rabbit and 2^nd secondary antibody is goat anti-rabbit, so goat serum should be used). Note: extra blocking step(s) may be needed to block some binding sites left open for the primary antibodies raised from the same species. Analyze each experiment individually.

13. 2^nd Primary Antibody: incubate sections in 2^nd primary antibody (rabbit) at appropriate dilution in antibody dilution buffer for 1 hour at room temperature.

14. Rinse in PBS-Tween 20 for 3x2 min.

15. 2^nd Secondary Antibody: incubate sections in 2^nd biotinylated secondary antibody – goat anti-rabbit (Vector Labs) in PBS for 20-30 minutes at room temperature.

16. Rinse in PBS-Tween 20 for 3x2 min.

17. 2^nd Detection: incubate sections in HRP-Streptavidin in PBSfor 20-30 minutes at room temperature. Note: don’t use serum (including BSA) solution to diluent HRP-Streptavidin since serum may contain biotin therefore competing Streptavidin binding with biotinylated secondary antibody, therefore reducing binding activity.

18. Rinse in PBS-Tween 20 for 3x2 min.

19. 2^nd Chromogen/Substrate: incubate sections in DAB-brown for 1-3 minutes to produce Brown reaction product.

20. Rinse in PBS-Tween 20 for 3x2 min.

21. Counterstain with methyl green if desired for 5 minutes at room temperature.

22. Rinse in distilled water briefly.

23.   Dehydrate quickly through 95% ethanol, 100% ethanol for 10 dips each.

24. Clear in xylene and coverslip with permanent mounting medium.

4. Results

• 1^st Primary Antibody Staining Sites -------------------- dark blue
• 2^nd Primary Antibody Staining Sites-------------------- brown
• Counterstained Nuclei ----------------------------------- light green

5. Notes

  This enzyme based double staining method is limited for the demonstration of two proteins at different locations. For example, one is nuclear protein, the other is cytoplasmic protein. If the two proteins are both nuclear or cytoplasmic or too close from each other, this method is not suitable.

One can also achieve different end point colors by using different chromogen substrates or by combining with alkaline phosphatase substrates.