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.

NOTE: Please see product datasheet or product webpage for appropriate antibody dilution, diluent 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. Wash Buffer:
    1. 1X Tris Buffered Saline with Tween® 20 (TBST): To prepare 1 L 1X TBST, add 100 ml 10X Tris Buffered Saline with Tween® 20 (TBST) (#9997) to 900 ml dH2O, mix.
  5. 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).
      • 1X PBST: To prepare 1 L 1X PBST, add 50 ml 20X Phosphate Buffered Saline with Tween® 20 (PBST) (#9809) to 950 ml dH2O, mix.
  6. Antigen Unmasking Options:
    1. 1X Citrate Unmasking Solution: To prepare 250 ml of 1X citrate unmasking solution, dilute 25 ml of SignalStain® Citrate Unmasking Solution (10X) (#14746) with 225 ml of dH2O.
    2. 1X EDTA Unmasking Solution: To prepare 250 ml add of 1X EDTA unmasking solution, dilute 25 ml of SignalStain® EDTA Unmasking Solution (10X) (#14747) with 225 ml of dH2O.
    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.
  7. 3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H2O2 to 90 ml dH2O.
  8. Blocking Solution: 1X TBST/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
    1. 1X TBST/5% Normal Goat Serum: add 250 µl Normal Goat Serum (#5425) to 5 ml 1X TBST.
    2. 1X Animal-Free Blocking Solution: to 4 mL of dH2O, add 1 ml of Animal-Free Blocking Solution (5X) (#15019).
  9. Detection System: SignalStain® Boost IHC Detection Reagents (HRP, Mouse #8125; HRP, Rabbit #8114).
  10. Substrate: SignalStain® DAB Substrate Kit (#8059).
  11. Hematoxylin: Hematoxylin (#14166)
  12. Mounting Medium: SignalStain® Mounting Medium (#14177)

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: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at sub-boiling temperature (95°-98°). Cool slides on bench top for 30 min.
  2. For EDTA: Heat slides submersed in 1X EDTA unmasking solution in a microwave until boiling is initiated; follow with 15 min at sub-boiling temperature (95°-98°). No cooling is necessary.
  3. For TE: Heat slides in a microwave submersed in 10 nM Tris/1 mM EDTA, pH 9.0 until boiling is initiated; follow with 18 min at sub-boiling temperature (95°-98°). Cool slides on bench top 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 of preferred 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.
  7. Incubate overnight at 4°C.
  8. Equilibrate SignalStain® Boost Detection Reagent to room temperature.
  9. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
  10. Cover section with 1-3 drops SignalStain® Boost Detection Reagent as needed. Incubate in a humidified chamber for 30 min at room temperature.
  11. Wash sections three times with wash buffer for 5 min each.
  12. Add 1 drop (30 µl) SignalStain® DAB Chromogen Concentrate to 1 ml SignalStain® DAB Diluent and mix well before use.
  13. Apply 100-400 µl SignalStain® DAB to each section and monitor closely. 1-10 min generally provides an acceptable staining intensity.
  14. Immerse slides in dH2O.
  15. If desired, counterstain sections with Hematoxylin (#14166) per instructions for use.
  16. Wash sections in dH2O two times for 5 min each.
  17. 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.
  18. Mount sections with coverslips and SignalStain® Mounting Medium (#14177).

posted February 2010

revised October 2016