Dario Siniscalco, Carlo Fuccio, Kevin A. Roth
The University of Alabama at Birmingham
Caspase-7 (CMH-1, Mch3, ICE-LAP3) has been identified as a major contributor to the execution of apoptosis. Caspase-7 is an effector caspase (along with caspase-2 and -3), meaning that it cleaves essential cellular machinery rather than activating other caspases. Caspase-7 is cleaved by many enzymes, including caspases-3, -6, -8, -9 and granzyme B. Once activated, caspase-7 cleaves many of the same substrates as caspase-3, including poly (ADP-ribose) polymerase, or PARP
Preparation of Slides:
Rats were perfused transcardially with 150 ml of saline solution (0.9% NaCl), and 250 ml of Bouin’s fixative (picric acid, 40% formaldehyde, acetic acid glacial). Lumbar spinal cord at the L4-L5 level was taken out and kept in the fixative for 24 hours followed by 75% ethanol for another 24 hours. The tissue was then dehydrated in graded ethanols and embedded in paraffin resin. Serial 5um sections of the lumbar segments of the spinal cord were sliced.
1. Deparaffinization: Sections were deparaffinized by further passages in Citric Solvent (Fisher Scientific, Pittsburgh, PA) and in Iso-propanol.
2. Antigen Retrieval: Citrate antigen retrieval was performed by placing slides in citrate buffer (0.1 M citric acid monohydrate, and 0.1 M sodium citrate, Sigma) in a water filled steamer for 20 min.
3. Blocking: After cooling, endogenous peroxidase activity was quenched by incubating sections for 15 min in 0.3% hydrogen peroxide in PBS and non-specific antibody binding was inhibited by incubation for 30 min in blocking solution (1% BSA, 0.2% powdered skim milk, 0.3% Triton-X 100 in PBS).
4. Primary Antibody: Primary antibodies were diluted in PBS blocking buffer and slides were incubated overnight at 4 C in primary antibodies to rabbit anti-caspase-7 (1:100; Cell Signaling, CA, Cat# 9491).
5. Secondary Antibody and Detection: HRP-labeled secondary antibodies (1:1,000; Jakson Immunoresearch Laboratories, West Grove, PA) specific to the IgG species used as a primary antibody and fluorescein labeled tyramide amplification (TSA; Perkin Elmer Life Science Products, Boston, MA) were used to locate the specific antigens in each section.
Sections were counterstained with bisbenzimide (Hoechst 33258) and
mounted with PBS:glycerol (1:1).
7. Observation: Fluorescently labelled sections were viewed with a Zeiss Axioskop (Zeiss, Germany) epifluorescence microscope and the images were captured with a Zeiss Axiocam and Axiovison software.
Figure 4. Caspases immunostaining. Caspase-7 immunostaining in lamina II spinal cord of rats 3 days after sciatic nerve chronic constriction injury (CCI). Strong immunohistochemical reactivity for caspase-7 was found in the dorsal horn cells of the spinal cord at 3 days after CCI (A: sham rat; B: CCI-rat). At 3 days post-CCI, 1-aminoindan-1,5-dicarboxylic acid (AIDA), a preferential metabotropic glutamate subtype 1 (mGlu1) receptor antagonist, treatment resulted in reduction staining for caspase-7 immunoreactivity in the superficial laminae of the spinal cord (Fig. C). Scale bar: 20 mm.