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: Please see 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 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 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).