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Immunohistochemical staining procedure (for manual procedure)

 We employ routine immunnostaining procedures for the demonstration of nuclear receptor localization in tissues without using specific secondary antibodies or buffer kits. Following is the procedure to stain formalin-fixed and paraffin-embedded specimens.

1. Four micron-thick sections should be used.
2. Dewax in xylol, 3 times x 5 min
3. Place the slides into glass slide chamber and fill them with the processing buffer
   (citric acid buffer, pH6.0-7.0, preferably pH6.0-6.5)
4. Antigen retrieval by autoclave (121℃, 15min).
5. Take out the glass slides in chamber, wait for 40 min (cooling down to about body temperature), and rinse
   the slides 3 times with distilled water or PBS by emptying and refilling the chambers.
6. Block endogenous peroxidase activity with freshly made 0.3% H2O2 in methanol, 20-30 min.
7. Three changes of PBS for five minutes. Preferably be treated with normal serum for 10 min. for blocking.
   (This blocking step may be skipped, if the second antibody works well.)
8. Incubate with monoclonal antibody, at 4℃, overnight.
9. Wash with PBS, 3 times x 5 min.
10. Incubate with second antibody, (for example, Simple Stain MAX-PO(MULTI) : NICHIREI BIOSCIENCE INC.),
   30-120 min., preferably 30-60 min. at room temperature.
11. Three changes of PBS for five minutes.
12. Stain with diaminobenzidin (DAB) solution, 10 min. at room temperature. 0.01% DAB in 0.05 M Tris/HCI
   (pH 7.4) solution should be filtrated. H2O2 must be added to a final concentration of 0.01%.
13. Wash with running tap water, 3 min.
14. Counterstain with Mayer's hematoxylin, 0.5 min.
15. Wash with running tap water.
16. Dehydrate with increasing solutions of ethanol: 50%, 70%, 96%, absolute, 3 min. each.
17. Clear with xylol, 3 times x 3 min.
18. Mount with mounting medium.

Revised by Shuying Jiang, Makoto Naito at Niigata University.
Oct. 24, 2012