Ethanol, anhydrous denatured, histological grade (100% and 95%)
Deionized water (dH2O)
Hematoxylin (optional)
Wash Buffer: 1X TBS/0.1% Tween-20 (1X TBST): To prepare 1 L add 100 ml 10X TBS to 900 ml dH2O. Add 1 ml Tween-20 and mix. 10X Tris Buffered Saline (TBS): (#9997) To prepare 1 L add 24.2 g Trizma base (C4H11NO3) and 80 g sodium chloride (NaCl) to 1 L dH2O. Adjust pH to 7.6 with concentrated HCl.
Antigen Unmasking: TE: 10 mM Tris/1 mM EDTA, pH 9.0: To prepare 1L add 1.21 g Trizma base (C4H11NO3) and 0.372 g EDTA (C10H14N2O8Na2•2H2O) to 950 ml dH2O. Adjust pH to 9.0, then adjust final volume to 1000 ml with dH2O.
3% Hydrogen Peroxide: To prepare, add 10 ml 30% H2O2 to 90 ml dH2O.
Blocking Solution: TBST/5% normal goat serum (#5425): to 5ml 1X TBST add 250 µl normal goat serum.
DAB Reagent or suitable substrate: Prepare according to manufacturer’s recommendations.
B. Deparaffinization/Rehydration
NOTE: Do not allow slides to dry at any time during this procedure.
Deparaffinize/hydrate sections:
Incubate sections in three washes of xylene for 5 minutes each.
Incubate sections in two washes of 100% ethanol for 10 minutes each.
Incubate sections in two washes of 95% ethanol for 10 minutes each.
Wash sections twice in dH2O for 5 minutes each.
C. Antigen Unmasking
Bring slides to a boil in 10 mM TE/1 mM EDTA, pH 9.0 then maintain at a sub-boiling temperature for 18 minutes. Cool on the bench for 30 minutes.
D. Staining
Wash sections in dH2O three times for 5 minutes each.
Incubate sections in 3% hydrogen peroxide for 10 minutes.
Wash sections in dH2O twice for 5 minutes each.
Wash section in wash buffer for 5 minutes.
Block each section with 100–400 µl blocking solution for 1 hour at room temperature.
Remove blocking solution and add 100–400 µl primary antibody diluted in recommended antibody diluent to each section. Incubate 30 minutes at room temperature.