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

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

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Immunohistochemistry Image 1: SignalStain® Apoptosis (Cleaved Caspase-3) IHC Detection Kit

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).

Immunohistochemistry Image 2: SignalStain® Apoptosis (Cleaved Caspase-3) IHC Detection Kit

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).

To Purchase # 12692S
Product # Size Price
12692S
1 Kit  (120 slides) N/A

Protocol

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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 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.

Background

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.

Pathways & Proteins

Explore pathways + proteins related to this product.

Limited Uses

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Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST's products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST's Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not For Use In Diagnostic Procedures.
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