*IMPORTANT: Please refer to the APPLICATIONS section on the front page of product datasheet or product webpage to determine whether a product is validated and approved for use on paraffin-embedded (IHC-P) tissue sections. Please see product datasheet or product webpage for appropriate antibody dilution and unmasking solution.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. Xylene.
  2. Ethanol, anhydrous denatured, histological grade (100% and 95%).
  3. Deionized water (dH2O).
  4. Hematoxylin (optional).
  5. Wash Buffer:
    1. 10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
  6. *Antibody Diluent Options:
    1. SignalStain® Antibody Diluent: (#8112).
    2. TBST/5% normal goat serum: To 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425).
    3. PBST/5% normal goat serum: To 5 ml 1X PBST, add 250 µl Normal Goat Serum (#5425).

      20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.

      1X PBS/0.1% Tween® 20 (1X PBST): To prepare 1 L 1X PBST, add 100 ml 10X PBS to 900 ml dH2O. Add 1 ml Tween® 20 and mix.

  7. *Antigen Unmasking Options:
    1. Citrate: 10 mM Sodium Citrate Buffer: To prepare 1 L, add 2.94 g sodium citrate trisodium salt dihydrate (C6H5Na3O7•2H2O) to 1 L dH2O. Adjust pH to 6.0.
    2. EDTA: 1 mM EDTA: To prepare 1 L add 0.372 g EDTA (C10H14N2O8Na2•2H2O) to 1 L dH2O. Adjust pH to 8.0.
    3. TE: 10 mM Tris/1 mM EDTA, pH 9.0: To prepare 1 L, add 1.21 g Tris base (C4H11NO3) and 0.372 g EDTA (C10H14N2O8Na2•2H2O) to 950 ml dH2O. Adjust pH to 9.0, then adjust final volume to 1 L with dH2O.
    4. Pepsin: 1 mg/ml in Tris-HCl, pH 2.0.
  8. 3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H2O2 to 90 ml dH2O.
  9. Blocking Solution: TBST/5% Normal Goat Serum: to 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425).
  10. Detection System: SignalStain® Boost IHC Detection Reagents (HRP, Mouse #8125; HRP, Rabbit #8114).
  11. Substrate: SignalStain® DAB Substrate Kit (#8059).

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 min each.
    2. Incubate sections in two washes of 100% ethanol for 10 min each.
    3. Incubate sections in two washes of 95% ethanol for 10 min each.
  2. Wash sections two times in dH2O for 5 min each.

C. Antigen Unmasking*

NOTE: Consult product datasheet for specific recommendation for the unmasking solution/protocol.

  1. For Citrate: Bring slides to a boil in 10 mM sodium citrate buffer, pH 6.0; maintain at a sub-boiling temperature for 10 min. Cool slides on bench top for 30 min.
  2. For EDTA: Bring slides to a boil in 1 mM EDTA, pH 8.0: follow with 15 min at a sub-boiling temperature. No cooling is necessary.
  3. For TE: Bring slides to a boil in 10 mM Tris/1 mM EDTA, pH 9.0: then maintain at a sub-boiling temperature for 18 min. Cool at room temperature for 30 min.
  4. For Pepsin: Digest for 10 min at 37°C.

D. Staining

NOTE: Consult product datasheet for recommended antibody diluent.

  1. Wash sections in dH2O three times for 5 min each.
  2. Incubate sections in 3% hydrogen peroxide for 10 min.
  3. Wash sections in dH2O two times for 5 min each.
  4. Wash sections in wash buffer for 5 min.
  5. Block each section with 100–400 µl blocking solution for 1 hr at room temperature.
  6. Remove blocking solution and add 100–400 µl primary antibody diluted in recommended antibody diluent to each section*. Incubate overnight at 4°C.
  7. Equilibrate SignalStain® Boost Detection Reagent to room temperature.
  8. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
  9. Cover section with 1–3 drops SignalStain® Boost Detection Reagent as needed. Incubate in a humidified chamber for 30 min at room temperature.
  10. Wash sections three times with wash buffer for 5 min each.
  11. Add 1 drop (30 µl) SignalStain® DAB Chromogen Concentrate to 1 ml SignalStain® DAB Diluent and mix well before use.
  12. Apply 100–400 µl SignalStain® DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
  13. Immerse slides in dH2O.
  14. If desired, counterstain sections with hematoxylin per manufacturer’s instructions.
  15. Wash sections in dH2O two times for 5 min each.
  16. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 sec each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 sec each.
    3. Repeat in xylene, incubating sections two times for 10 sec each.
  17. Mount sections with coverslips.

posted February 2010

revised November 2013

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