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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded E14 mouse embryo using Cleaved Caspase-3 (Asp175) (D3E9) Rabbit mAb #9579 (left) or Rabbit (DA1E) mAb IgG XP® SignalStain® Isotype Control #12960 (right).

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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded Kelly xenograft using Cleaved Caspase-3 (Asp175) (D3E9) Rabbit mAb #9579 (left) or Rabbit (DA1E) mAb IgG XP® SignalStain® Isotype Control #12960 (right).

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Immunohistochemistry (Paraffin)

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. 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.
  8. 3% Hydrogen Peroxide: To prepare, add 10 ml 30% H2O2 to 90 ml dH2O.
  9. Blocking Solution: TBST/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
    1. TBST/5% Normal Goat Serum: to 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425).
    2. 1X Animal-Free Blocking Solution: to 4 mL of dH2O, add 1 ml of Animal-Free Blocking Solution (5X) (#15019).
  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. For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). 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 of preferred 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 (#3900) 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 with hematoxylin (#14166).
  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 sections with coverslips and mounting medium (#14177).

posted July 2013

revised June 2016

Protocol Id: 64
Product Includes Quantity Storage Temp
Cleaved Caspase-3 (Asp175) (D3E9) Rabbit mAb 9579 100 µl -20°C
Rabbit (DA1E) mAb IgG XP® SignalStain® Isotype Control 100 µl -20°C
Normal Goat Serum 5425 2.4 ml -20°C
SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) 8114 12 ml 4°C
SignalStain® DAB Diluent 12 ml 4°C
SignalStain® DAB Chromogen Concentrate 360 µl 4°C
Tris Buffered Saline with Tween® 20 (TBST-10X) 9997 2.4 ml RT
SignalStain® Antibody Diluent 8112 25 ml 4°C

Product Description

SignalStain® Apoptosis (Cleaved Caspase-3) IHC Detection Kit allows the detection of activated caspase-3 in formalin-fixed paraffin-embedded human and mouse tissue samples. Cleaved Caspase-3 (Asp175) (D3E9) Rabbit mAb is detected by the polymer based, HRP-conjugated SignalStain® Boost IHC Detection Reagent in combination with SignalStain® DAB Diluent and Chromogen Concentrate. Also included is a concentration-matched rabbit monoclonal IgG control to verify the specificity of staining.

This combination of reagents provides a sensitive and specific means of detecting apoptotic events in tissue samples.


Specificity / Sensitivity

SignalStain® Apoptosis (Cleaved Caspase-3) IHC Detection Kit detects endogenous levels of the activated caspase-3 large fragment (17/19 kDa) resulting from cleavage adjacent to Asp175. This antibody does not recognize full-length caspase-3 or other cleaved caspases. This kit was developed for and is recommended for immunohistochemistry only.


Species Reactivity: Human, Mouse

Source / Purification

Cleaved Caspase-3 (Asp175) (D3E9) Rabbit mAb is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Asp175 of human caspase-3 protein. Rabbit (DA1E) mAb IgG XP® SignalStain® Isotype Control is not directed against any known antigen. It functions as an isotype control for rabbit IgG monoclonal antibodies.

Caspase-3 (CPP-32, Apoptain, Yama, SCA-1) is a critical executioner of apoptosis, as it is either partially or totally responsible for the proteolytic cleavage of many key proteins, such as the nuclear enzyme poly (ADP-ribose) polymerase (PARP) (1). Activation of caspase-3 requires proteolytic processing of its inactive zymogen into activated p17 and p12 fragments. Cleavage of caspase-3 requires the aspartic acid residue at the P1 position (2).


1.  Fernandes-Alnemri, T. et al. (1994) J Biol Chem 269, 30761-4.

2.  Nicholson, D. W. et al. (1995) Nature 376, 37-43.


Entrez-Gene Id 836
Swiss-Prot Acc. P42574


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
SignalStain is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
Trizma is a registered trademark of Sigma-Aldrich Co. LLC.
Tween is a registered trademark of ICI Americas, Inc.

12692
SignalStain® Apoptosis (Cleaved Caspase-3) IHC Detection Kit