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1) Heat to approx 600C. Add 1 small drop 10 N NAOH. Solution will begin to turn clear after 5 min. Titrate pH to approx 7.8. Cool to RT before use.
2) MeOH/H202 (0.45%): 200 ml 100% MEOH
3 ml 30% H202
Use immediately!
3) Blocking SERUM/PBS:
A) For monoclonals 3.3 ml PBS
1 drop (45 ul) serum (1.3%)
B) For polyclonals 0.6 ml PBS
0.3 ml 10% BSA/PBS
3% BSA, 10% serum 0.1 ml serum
4) Primary antibody dilution made in SERUM/PBS (in 3,4 use serum from same species as secondary antibody):
5 ml PBS
1 drop (45 ul) serum
5) SECONDARY ANTIBODY; 10 ul biotinylated antibody (from Vector Laboratories) 1.99 ml of SERUM/PBS
6) ABC REAGENT; (Vector Laboratories)
i) 10 ul A in 1 ml PBS. Vortex lightly.
ii) 10 ul B in A/PBS mixture. Vortex.
iii) Let stand at room temp for 30 min. before using.
7) DAB/H202 SOLUTION: PREPARE JUST PRIOR TO USEI!!
(DAB 3,31-diaminobenzidine tetrahydrochloride, Sigma D 5637)
0.05% DAB
0.01% H202
0.05 M TRIS pH 7.2
i) Prepare up stock 1OX DAB solution (0.5%) in 0.05 M TRIS
pH 7.2 and store in 15 ml aliquots at -20 C. For example, dissolve 750 mg diaminobenzidine tetrahydrochloride in 150 ml of tris buffer, and store ten aliquots for future use.
ii) To prepare 150 ml DAB bath: thaw 15 ml aliquot of 1OX " DAB and place on ice. Immediately prior to use:
15 ml 1OX DAB
135 ml 0.05 M TRIS pH 7.2
0.05 ml of 30% H202
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