Rabbit anti cleaved caspase 3 asp175 antibody

Dissection and staining protocol were reported previously (Deng et al., 2015 (link)). In brief, intact guts were fixed at room temperature for 45 min in 100 mM glutamic acid, 25 mM KCl, 20 mM MgSO4, 4 mM sodium phosphate, 1 mM MgCl2, 4% formaldehyde. All subsequent incubations were done in PBS, 0.5% BSA, 0.1% TritonX-100 at 4°C. rabbit anti-pH3 (Y408884, Applied Biological Materials Inc.) 1:500, rabbit anti-cleaved-caspase-3 (Asp175 Antibody, #9661, Cell Signaling Technology) 1:200. Fluorescent secondary antibodies were obtained from Jackson ImmunoResearch. DAPI was used to stain DNA.

A time course immunocytochemistry study of caspase-3 expression was carried out for A/J and B6 mice (four for each group at each time point). The time points selected were 2, 4, 10, and 46 weeks. Paraffin sections (5 µm) were made, as described previously (Han et al., 2012 (link)), except that the inner ears were fixed in 4% paraformaldehyde and decalcified with 10% EDTA. After dewaxing, the sections were subjected to heat-induced antigen retrieval in citrate buffer (0.01 mol/L, PH 6.0), washed in 1 × PBS at room temperature thrice for 5 min, and permeabilized in 0.5% Triton X-100 for 15 min. After being washed thrice in 1 × PBS for 5 min and blocked in 3% goat serum and 2% bovine serum albumin at 37℃ for 1 hr, the samples were immersed in rabbit anticleaved caspase-3 (Asp175) antibody (1:400 dilution) (Cell Signaling Technology, Inc., Danvers, MA, USA) and incubated at 4℃ overnight. Samples with primary antibody omitted were used as the negative controls. After being washed thrice in 1 × PBS for 5 min, the samples were immersed in anti-rabbit secondary antibody- Alexa 488 (1:800 dilution) at 37℃ for 1 hr, and then counterstained with Hoechst33342 (10 µg/ml in PBS) for 15 min at room temperature. Finally, the sample mounts were observed under immunofluorescent confocal microscopy (Leica DMI4000 B, Leica Microsystems, Wetzlar, Germany).

Xenograft tumour tissue was cryo-sectioned (20 µm) and processed as previously described.^{6 (link)} Endothelial cells were stained with a monoclonal Armenian-hamster anti-mouse CD31 antibody (1:200, Abcam) or a fluorescein isothiocyanate (FITC)-labelled anti-CD31 antibody (1:200, clone 390, Biolegend), and apoptotic cells were detected with a rabbit anti-cleaved caspase-3 (Asp175) antibody (1:200, Cell Signaling). Pericytes were detected either by a rabbit anti-NG2 Chondroitin Sulphate Proteoglycan antibody (1:100, Millipore) or a rat anti-PDGF-R antibody (1:100, CD140b, eBioscience). Vascular endothelial (VE) cadherin was stained with a rat anti-CD144 (VE-cadherin) antibody (BD Pharmingen). The following secondary antibodies derived from goat were used to label the respective primary antibodies with fluorescent dyes: Alexa Fluor 647 anti-hamster, Alexa Fluor 594 anti-rat, Alexa Fluor 594 anti-rabbit and Alexa Fluor 488 anti-hamster (1:500, Thermo Fisher Scientific). Vascular leakage was quantified by analysing intravenously injected FITC-dextran (molecular weight 2000 kDa, Sigma), pimonidazole adducts were detected with a FITC-labelled anti-pimonidazole antibody to visualise hypoxia; both procedures have been previously described in detail^{6 (link),19 (link)}; nuclei were stained with 4,6-diamidin-2-phenylindol (DAPI).