A. Solutions and Reagents

  1. Xylene
  2. Ethanol, anhydrous denatured, histological grade (100% and 95%)
  3. Deionized water (dH2O)
  4. Hematoxylin (optional)
  5. 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.
  6. Antibody Diluent: SignalStain® Antibody Diluent (#8112)
  7. 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.
  8. 3% Hydrogen Peroxide: To prepare, add 10 ml 30% H2O2 to 90 ml dH2O.
  9. Blocking Solution: TBST/5% normal goat serum (#5425): to 5ml 1X TBST add 250 µl normal goat serum.
  10. SignalStain® Boost IHC Detection Reagent (HRP, Mouse) (#8125).
  11. 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.

  1. Deparaffinize/hydrate sections:
    1. Incubate sections in three washes of xylene for 5 minutes each.
    2. Incubate sections in two washes of 100% ethanol for 10 minutes each.
    3. Incubate sections in two washes of 95% ethanol for 10 minutes each.
  2. Wash sections twice in dH2O for 5 minutes each.

C. Antigen Unmasking

  1. 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

  1. Wash sections in dH2O three times for 5 minutes each.
  2. Incubate sections in 3% hydrogen peroxide for 10 minutes.
  3. Wash sections in dH2O twice for 5 minutes each.
  4. Wash section in wash buffer for 5 minutes.
  5. Block each section with 100–400 µl blocking solution for 1 hour at room temperature.
  6. Remove blocking solution and add 100–400 µl primary antibody diluted in recommended antibody diluent to each section. Incubate 30 minutes at room temperature.
  7. Equilibrate SignalStain® Boost IHC Detection Reagent (HRP, Mouse) (#8125) reagent to room temperature.
  8. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
  9. Add 1–2 drops of SignalStain® Boost IHC Detection Reagent (HRP, Mouse) (#8125) to each section. Incubate 30 minutes at room temperature.
  10. Remove SignalStain® Boost IHC Detection Reagent (HRP, Mouse) (#8125) and wash sections three times with wash buffer for 5 minutes each.
  11. Add 100–400 μl DAB or suitable substrate to each section and monitor staining closely.
  12. Upon completion of development, immerse slides in dH2O.
  13. If desired, counterstain sections in hematoxylin per manufacturer’s instructions.
  14. Wash sections in dH2O two times for 5 minutes each.
  15. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 seconds each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 seconds each.
    3. Repeat in xylene, incubating sections two times for 10 seconds each.
  16. Mount coverslips.

posted November 2010

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