Rabbit anti caspase 3

Immediately after sacrifice, the hearts were excised, washed, and fixed in 10% neutral buffered formalin for 24 hr. Five μm-thick paraffin sections were prepared, cut, and stained with hematoxylin and eosin (H&E). Other sections were stained with Masson's trichrome. Stained heart sections were scanned and examined using light microscopy.
Other heart sections were blocked via incubation in 3% hydrogen peroxide and washed in Tris-buffered saline (TBS; pH 7.6). The slides were incubated with protein block (Novocastra) to prevent nonspecific binding of antibodies and probed with rabbit anti-caspase-3 (Santa Cruz Biotechnology, USA). The sections were then probed with anti-rabbit secondary antibody, washed, and counterstained with hematoxylin. Negative control sections were similarly processed with omission of incubation with the primary antibody.

Homogenate samples with a volume of 65 μL were mixed with 25 μL NuPAGE LDS 4× sample buffer and 10 μL reducing agent (ThermoFisher Scientific) and heated at 70 °C for 10 min. Individual samples were run on 4–12% NuPAGE Bis-Tris gels (Novex) and transferred to reinforced nitrocellulose membranes. After blocking with 30 mM Tris-HCl (pH 7.5), 100 mM NaCl and 0.1% Tween 20 containing 5% fat-free milk powder for 60 min at RT, the membranes were incubated with the rabbit anti-actin (1:200; Sigma) or rabbit anti-caspase3 (1:1000; Santa Cruz) primary antibodies at RT for 60 min. After washing, membranes were incubated with a peroxidase-labeled goat anti-rabbit secondary antibody (1:2000, Vector Laboratories) for 30 min at RT. Immunoreactive species were visualized using the Super Signal West Dura substrate (ThermoFisher Scientific) and an LAS 3000 cooled CCD camera (Fujifilm).

The muscles were homogenized in Tris-EDTA buffer with a cocktail of protease inhibitors and 1 mM phenylmethanesulfonyl fluoride. Proteins were subjected to SDS-PAGE, transferred onto polyvinylidene difluoride membranes (Millipore, USA), and probed with mouse anti-MHC (1 : 1,000) (MF-20, Developmental Studies, Hybridoma Bank, University of Iowa, USA), mouse anti-tubulin (1 : 5,000), mouse anti-GAPDH (1 : 5,000), rabbit anti-Bax (1 : 500), rabbit anti-Bcl2 (1 : 500), rabbit anti-caspase-3 (1 : 500), rabbit anti-Ub proteins (1 : 500) (Santa Cruz Biotechnology, USA), rabbit anti-LC3B (1 : 1,500) (Cell Signaling, USA), and rabbit anti-p62 (1 : 1,500) (Abcam, USA). All immunoreactions were visualized by enhanced chemiluminescence (Thermo Scientific, USA). Images were acquired using Fotodyne FOTO/Analyst Luminary Workstation Systems (Fotodyne, Inc., USA).

Double fluorescence labeling was performed as described previously [30 (link)]. Amongst the stored brains after India Ink angiography, 12 brains were randomly used from groups sham (n=3), SAH (n=3), low atorvastatin group (n=3), and high atorvastatin group (n=3), respectively. The intracranial internal carotid artery (ICA) was sectioned every 200 μm. Ten micrometers thick coronal sections were cut by a cryostat and incubated overnight at 4°C with the rabbit anti-Caspase 3 (1:50; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-CHOP (1:500; Sigma-Aldrich, St. Louis, MO) antibodies, followed by incubation with appropriate fluorescence dye-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA). The sections were visualized with a fluorescence microscope, and pictographs were recorded and analyzed with MagnaFire SP 2.1B software (Olympus, Melville, NY).

Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. For detection of proteins, membranes were incubated with antibodies diluted in 5% milk powder in Tris-buffered saline solution containing 0.1% Tween-20. Antibodies against phospho-groups were diluted in 5% BSA instead of milk powder. The following primary antibodies were used: goat anti-G2E3, mouse anti-hsc70, mouse anti-p53 (DO-1) (all Santa Cruz Biotechnology), rabbit anti-Caspase 3, rabbit anti-cleaved Caspase 3, mouse anti-Chk1, rabbit anti-PARP, rabbit anti-phospho-Chk1 (Ser317), rabbit anti-phospho-Chk2 (Thr68) (all Cell Signaling Technology), mouse anti-Chk2, mouse anti-Mdm2 (Ab-1), mouse anti-p21, mouse anti-PARP (all Calbiochem), mouse anti-actin (AC-15), rabbit anti-IgG (all Abcam), mouse anti-phospho-H2AX (Ser319) (Millipore), mouse anti-HA-tag (16B12, Covance), mouse anti-GFP (Clontech). Primary antibodies were detected by peroxidase-coupled secondary antibodies (Jackson ImmunoResearch Europe) using a Chemoluminescence Imaging System (Intas).

Proteins were separated on SDS-PAGE (8-12%) gels and
transferred to PVDF membranes (Millipore, Germany).
Membranes were blocked with 5% non-fat milk in PBS0.1%
TweenTM20 and were incubated overnight at 4°C
with the respective specific primary antibody for each
protein [mouse anti-LRSAM1/Abcam ab73113 (1:400),
rabbit anti-LRSAM1/Novus Biological H00090678-D01
(1:750), rabbit anti-CASPASE-3/Santa Cruz SC7148
(1:700), mouse anti-CYCLIN D1/Abcam ab6152 (1:300),
mouse anti-TSG101/Novus Biological NB200-11 (1:500)
and mouse anti-ß-ACTIN/Sigma-Aldrich A2228 (1:4000)]
which was diluted in phosphate buffered saline (PBS)-0.1%
TweenTM20. The membranes were then incubated for 2 hours
with the appropriate secondary antibodies [AP124P goat
anti-mouse IgG-Peroxidase H+L/Millipore (1:7000) and
sc-2077 donkey anti-rabbit IgG-HRP/ Santa Cruz (1:7000)]
followed by incubation with the visualization LumiSensorTM
Chemiluminescent HRP Substrate Kit (Genscript, USA).
Membranes were finally visualized using the UVP imaging
system (BioRad, USA). Western blots were quantified using
the ImageJ software (https://imagej.nih.gov). Protein quantity
ratio was estimated relative to ß-actin.