Upstream / Downstream

Explore pathways related to this product.

Antibody Guarantee

CST Antibody Performance Guarantee

LEARN MORE  

Questions?

Find answers on our FAQs page.

ANSWERS  

Visit PhosphoSitePlus®

PTM information and tools available.

LEARN MORE

We recommend the following alternatives

          H M
Image
Image

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).

Learn more about how we got this image

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).

Learn more about how we got this image
Image
Image
Image
Image
Page

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. Antigen Unmasking: 10 mM Sodium Citrate Buffer: To prepare 1 L, add 2.94 g sodium citrate trisodium salt dihydrate (C6H5Na3O72H2O) 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).

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 (#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 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.

posted July 2013

protocol id: 64

Product Includes Quantity Isotype
Cleaved Caspase-3 Blocking Peptide 1 x 0.1 ml  
Peroxide Quench 1 x 19.5 ml  
Blocking Solution 1 x 15 ml  
Prediluted Negative Control 1 x 15 ml  
Prediluted Cleaved Caspase-3 (D175) Ab #9661 1 x 15 ml  
Biotinylated Secondary Ab 1 x 15 ml  
Reagent A 1 x 850 µl  
Reagent B 1 x 850 µl  
NovaRED Substrate 1 (TM) 1 x 600 µl  
NovaRED Substrate 2 (TM) 1 x 600 µl  
NovaRED Substrate 3 (TM) 1 x 600 µl  
NovaRED Substrate 4 (TM) 1 x 800 µl  
Mixing Bottle 1 x    

Order Details

Custom Ordering Details: The control slides that accompany this kit are cut freshly upon ordering. Please allow three to five days for your order to be processed and shipped.

Product Description

CST’s Apoptosis Marker: SignalStain® Cleaved Caspase-3 (Asp175) IHC Detection Kit is a "ready to use" system designed to detect the activation of caspase-3 in human tissue and cell preps by immunohistochemistry. The kit utilizes the ABC immunoperoxidase method to detect endogenous levels of caspase-3 protein. Prediluted Cleaved Caspase-3 (Asp175) Antibody is bound by a biotinylated secondary antibody. Avidin DH and biotinylated horseradish peroxidase are complexed by mixing defined amounts prior to use, and the mixture subsequently binds the secondary antibody. The macromolecular complex is localized by incubation with NovaRED™ enzyme substrate.

The prediluted primary antibody, along with the ABC system, allows the user to examine caspase-3 localization consistently and offers the highest sensitivity with the lowest background.


Specificity / Sensitivity

Apoptosis Marker: SignalStain® Cleaved Caspase-3 (Asp175) IHC Detection Kit detects endogenous levels of the large fragment (17/19 kDa) of activated caspase-3 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 predicted to react based on 100% sequence homology: Rat

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to amino-terminal residues surrounding to Asp175 in human caspase-3. Antibodies are purified by protein A and peptide affinity chromatography.

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, 30761-30764.

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.
NovaRED™ is a trademark of Vector Laboratories.