Immunohistochemistry Protocol - Specific For Product: 12692

Specific For: SignalStain^® Apoptosis (Cleaved Caspase-3) IHC Detection Kit #12692

A. Solutions and Reagents

1. Xylene.
2. Ethanol, anhydrous denatured, histological grade (100% and 95%).
3. Deionized water (dH₂O).
4. Hematoxylin (optional).

5. Wash Buffer.

   NOTE: Additional 10X TBST will be required for washes.

   1. Tris Buffered Saline with Tween^® 20 (TBST-10X) #9997: To prepare wash buffer add 100 μl of Tris Buffered Saline with Tween^® 20 (TBST-10X) #9997 to 900 ml of dH₂O. OR
   2. 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. 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.
6. Antibody Diluent: SignalStain^® Antibody Diluent #8112
7. Antigen Unmasking: 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.
8. 3% Hydrogen Peroxide: To prepare, add 10 ml 30% H₂O₂ to 90 ml dH₂O.
9. Blocking Solution: 1X TBST/5% normal goat serum: Add 100 μl 10X TBST (#9997) and 50 μl normal goat serum (#5425) to 850 μl dH₂O.
10. Detection System: SignalStain^® Boost IHC Detection Reagent (HRP, Rabbit) (#8114).
11. Substrate: SignalStain^® DAB Substrate Kit (#8059), which includes SignalStain^® DAB Diluent (#11724) and SignalStain^® DAB Chromogen Concentrate (#11725).
12. Hematoxylin: Hematoxylin (#14166).
13. 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 twice in dH₂O for 5 min each.

C. Antigen Unmasking

1. Bring slides to a boil in 10 mM sodium citrate buffer pH 6.0, then maintain at a sub-boiling temperature for 10 min. Cool slides on bench top for 30 min.

D. Staining

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 section in wash buffer for 5 min.
5. Block each section with 100–200 μl blocking solution for 1 hr at room temperature.
6. Remove blocking solution and add 100–200 μl primary antibody diluted at 1:500 in SignalStain^® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
   1. Rabbit (DA1E) mAb XP^® SignalStain^® Isotype Control (#12960) is used as a negative control. Dilute at 1:500 in SignalStain^® Antibody Diluent (#8112) and apply 100–200 μl to each section.
7. Equilibrate SignalStain^® Boost Detection Reagent (#8114) 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 (#8114) 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 30 μl SignalStain^® DAB Chromogen Concentrate (#11725) to 1 ml SignalStain^® DAB Diluent (#11724) 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.