Immunohistochemistry Protocol – Paraffin for SignalStain^® Boost Detection Reagent

*IMPORTANT: See product datasheet for the appropriate antibody diluent, dilution, and antigen unmasking procedure.

A. Solutions and Reagents

NOTE: Prepare solutions with purified water.

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): 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:
   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 PBS/0.1% Tween-20 (1X PBST): To prepare 1 L, add 100 ml 10X PBS to 900 ml dH₂0. Add 1 ml Tween-20 and mix.

10X Phosphate Buffered Saline (PBS): To prepare 1 L, add 80 g sodium chloride (NaCl), 2 g potassium chloride (KCl), 14.4 g sodium phosphate, dibasic (Na₂HPO₄) and 2.4 g potassium phosphate, monobasic (KH₂PO₄) to 1 L dH₂O. Adjust pH to 7.4.

7. *Antigen Unmasking:
   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 Trizma^® base (C₄H₁₁NO₃) and 0.372 g EDTA (C₁₀H₁₄N₂0₈Na₂) 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, 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 (mouse #8125, 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 twice in dH₂O for 5 min each.

C. Antigen Unmasking*

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

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 twice 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 in 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 minutes generally provides an acceptable staining intensity.
13. Immerse slides in dH₂O.
14. If desired, counterstain sections in 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 coverslips.