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Active Caspase-3 and-7 Immunolabeling

IHC detection of apoptosis-related proteins was carried out on 5-μm-thick deparaffinized sections. Before IHC, sections were subjected to heat-induced epitope retrieval by incubation in a 0.01 M sodium citrate solution (pH 6) at 120C for 10 min, followed by a 2-hr cool-down. Primary antibodies were diluted in the following buffer: 0.1 M PBS, 0.3% (m/v) BSA, 0.1% (m/v) sodium azide, 0.06% (m/v) n-ethyl-maleimide, and 20% (v/v) glycerol (PAB). Active caspase-3 was detected with a species-unspecific rabbit polyclonal antibody (1:1000 diluted; BD Biosciences, Le Pont-de-Claix, France) that specifically recognize the large fragment (17 kDa) of the active protein but not full-length caspase-3. The large fragment of active caspase-7 (20 kDa) was targeted by a species-unspecific rabbit polyclonal antibody (1:50 diluted) provided by Cell Signaling (Danvers, MA). For c-PARP IHC, sections were incubated in a solution of a rabbit polyclonal antibody (1/25 diluted; Cell Signaling, Danvers, MA). This species-specific antibody detects endogenous levels of the large fragment (89 kDa) of human PARP-1 resulting from cleavage at aspartic acid 214 but does not recognize full-length PARP-1 or other PARP isoforms. All antibodies applied in this study were previously used in Western blot analysis with satisfying results (Marchal et al. 2005,2007). Primary antibodies were applied for 16 hr at 4C. The sections were washed in two changes of PBST (0.1 M phosphate buffer, pH 7.4, 0.1% (v/v) Tween 20) over 10 min and incubated with biotinylated goat anti-rabbit antibody (Dakocytomation; Trappes, France) diluted 1:200 for 1 hr at room temperature. After two PBST washes (for 10 min each), endogenous peroxidase activity was blocked by a 10-min incubation in a 6% hydrogen peroxide solution in distilled water. The slides were washed two times in PBST (for 5 min each time) and incubated in streptavidin-peroxidase (Dakocytomation) diluted 1:150 in PBST for 1 hr at room temperature. After two PBST washes of 5 min, bound peroxidase was identified using the Novared TR system (Abcys; Paris, France). Nuclear counterstaining was performed with 1/2-diluted Harris hematoxylin. Each assay was controlled negatively by processing sections in the absence of primary antibody. Good immunostaining data (i.e., low background staining and good precision) were obtained with three antibodies. False-positive cells were only observed in tissue sections of tumor transplants where endogenous biotin was detected by the streptavidin–biotin system of visualization. However, it was easy to distinguish between positive, negative, and false-positive cells. Tumor cells were never artifactually stained, and false-positive staining was limited to mastocytes in mouse connective tissue around the tumors and in sebaceous glands when present.

Article Assessment of Apoptosis by Immunohistochemistry to Active Ca...

Apoptosis Experiments

For long-term apoptosis experiments, KB cells in T-75 flasks were treated with either ricin (1 ng/ml) or a mixture of tumor necrosis factor (TNF; 10 ng/ml) together with cycloheximide (5 μg/ml) for 2 days. The floating cells from treated flasks were then harvested at room temperature (RT) using a Shandon Cytospin centrifuge, fixed in 3.7% formaldehyde for 15 min, and permeabilized using 0.1% Triton X-100 for 5 min. The cytospins were then incubated in rabbit anti-cleaved caspase 3 antibody (1:100 dilution of manufacturer's stock) in 1% bovine serum albumin with PBS for 30 min. After a PBS wash, the cells were incubated with 25 μg/ml affinity-purified goat anti-rabbit IgG conjugated with rhodamine for 30 min. After washing and postfixation in formaldehyde, the preparations were incubated with 0.1 μg/ml DAPI in methanol and mounted under a coverslip in glycerol.

For shorter-term apoptosis experiments, two T-75 flasks of KB cells were incubated with 1 μg/ml ricin for 7 hr, and the floating cells were removed by shaking and pelleted by centrifugation at 5000 × g in 1% BSA-PBS. Similarly, untreated control cells were removed from flasks by trypsinization and pelleted in 1% BSA-PBS. Small aliquots of these preparations before centrifugation were also used to prepare cytopreps that were fixed in acetone and stained with hematoxylin. The pellets were fixed by slowly dripping 3.7% formaldehyde in PBS on top of the solid pellets and incubating overnight at RT. The pellets were then gently removed from the centrifuge tubes, placed in filter paper and into a cassette, and routinely processed for paraffin embedding.

Immunohistochemistry using the rabbit anti-cleaved caspase 3 antibody was performed on deparaffinized tissue sections using a routine avidin-biotin-immunoperoxidase technique [Vectastain Elite avidin-biotin-immunoperoxidase kit reagents (Vector Labs, Burlingame, CA)]. Before incubation with the primary antibody (1:200 dilution as supplied by the manufacturer), tissue sections were subjected to heat-induced epitope retrieval by incubation in a pH 8.0 0.01 M EDTA solution for 10 min in a vegetable steamer, followed by 20-min cool-down and treatment with 3% hydrogen peroxide before antibody application.

Article Improved Detection of Apoptotic Cells in Archival Paraffin S...

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Read this article:

Detection of Apoptosis in Paraffin Embedded Tissues: the Influence of Tissue Type and Fixation .

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Thank you Sumia and Alexandr. I will check those articles.