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Immunohistochemistry Protocol - Paraffin for SignalStain® Boost Detection Reagent

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: Consult product-specific protocol 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; HRP, Rat #72838; HRP, Goat #63707; AP, Mouse #31926; AP, Rabbit #18653; AP, Rat #15764; AP, Goat #26927).
  10. Substrate: HRP compatible: SignalStain® DAB Substrate Kit (#8059); SignalStain® Vivid Purple Peroxidase Substrate Kit (#96632); AP compatible: SignalStain® Vibrant Red Alkaline Phosphatase Substrate Kit (#76713)
  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-specific protocol 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 mM 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-specific protocol 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. Prepare the substrate as recommended in the Product Usage Information.
  13. Apply 100-400 µl substrate to the slides. Monitor the reaction. The recommended reaction time differs depending upon the substrate used. Refer to the Product Usage Information for specific guidance.
  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 January 2022