Anti caspase 3 antibody

EPC cells were cultured with M199 (Procell, USA) culture medium containing 10% FBS (Everygreen, Hangzhou, China) in a 25 cm² culture flask. SCRV virus was stored in a −80 °C refrigerator in our laboratory. The virus titer (TCID₅₀) was measured in a 96-well plate using the traditional Reed–Muench method.
3-Methyladenine (3-MA), rapamycin (RAPA) and Z-Val-Ala-Asp(OMe)-FMK (Z-VAD (OME)-FMK) were all obtained from MedChemExpress (MCE, New Jersey, USA). Apoptosis inducer was bought from Biyuntian Co. Ltd. (Shanghai, China). The Annexin V-FITC/PI detection Kit was acquired from Yeasen Biotech Co., Ltd. A TUNEL detection Kit (IV-CY3) was purchased from Boster Biological Technology Co. Ltd. (Wuhan, China). Anti-LC3B, anti-p62, anti-GAPDH and secondary antibodies were purchased from Sigma (Shanghai, China), and anti-caspase-3 antibody was purchased from Proteintech Group, Inc. The reverse transcription kit (Hifair II 1st Strand cDNA Synthesis SuperMix for qPCR) used in this study was bought from Vazyme Biotech Co., Ltd. (Nanjing, China).

Primary mouse neuron/glia cultures were infected with JEV and treated with MK-801, then cells were fixed in 4% formaldehyde for 10 min and blocked with 10% bovine serum albumin (BSA) in PBS for 30 min. Afterward, cells were stained with rabbit polyclonal anti-caspase-3 antibody (Proteintech) and mouse monoclonal anti-neuronal nuclei (NeuN) antibody (Chemicon) for 1 h. Then cells were washed three times with PBS and incubated with Alexa Fluor 488- and 555-conjugated secondary antibody (Invitrogen) for 30 min. The cell nuclei were stained with DAPI (Invitrogen). The staining results were observed using a fluorescence microscope (Zeiss).

PASMCs were cultured on sterile glass coverslips in 24-well plates and washed with cold TBST 4 times. Cells were fixed with 4% paraformaldehyde for 15 minutes. Then, the cell membrane was permeabilized with 0.3% Triton X-100 for 1 hour and blocked with normal goat serum for 1 hour at 37 °C. Cells were incubated with an anti-α-SMA antibody (4 µg/mL; BOSTER), an anti-Caspase-1 antibody (10 µg/mL; BOSTER), an anti-Caspase-3 antibody (5.4 µg/mL; Proteintech), an anti-Caspase-9 antibody (4 µg/mL Beyotime Biotechnology, Shanghai, China), anti-Ki-67 antibody (10 µg/mL; BOSTER), anti-Cleaved Caspase-3 (5 µg/mL;Cell Signaling Technology), and an anti-IL-18 antibody (7.6 µg/mL; Proteintech) overnight at 4 °C and were subsequently incubated with FITC-conjugated goat anti-rabbit and Cy3-conjugated goat anti-mouse antibodies for 2 hours at 37 °C. Cells were then washed with TBST, and nuclei were stained with DAPI (Beyotime Biotechnology, Shanghai, China) for 15 minutes at room temperature. ImageJ was used to detect mean gray value, the mean gray value (mean)=integrated density (IntDen)/area, 3 to 5 pictures and 20 to 40 individual cells were measured in different regions of the stained sections. For background correction, we used the default threshold to eliminate the error caused by manually selecting the threshold.

Liver cancer tissue array (No. 08C03), comprising 149 liver tumors, was purchased from Xi'an Aomei Biotechnology (Xi'an, China). Breast cancer tissue were immediately obtained from Tianjin Cancer Hospital (Tianjin, China) after surgical resection. IHC staining assay was performed as described previously [8 (link)]. The slides were incubated with rabbit anti-C4BPα antibody (1:250 dilution Proteintech Group, Chicago, USA), anti-Ki67 antibody (1:200 dilution, Proteintech Group, Chicago, USA), anti-caspase3 antibody (1:200 dilution, Proteintech Group, Chicago, USA) at 4°C for overnight. Then, the slides were incubated with horseradish peroxidase labelled secondary antibody for 30 min at room temperature. Immunostaining was developed by using chromogen 3, 3′-Diaminobenzidine (DAB), and counterstained with Mayer's hematoxylin (ZSBG-BIO, Beijing, China). The staining levels of C4BP were classified into three groups using a modified scoring method based on the intensity of staining (− negative; + low; ++Middle; +++ high; ++++super high).

The chemicals used in our experiments were: Sempervirine (Percent Purity, ≥98.0%; ChemFaces, CFS201902), MTT (Sigma-Aldrich, M2128), z-VAD-FMK (Apexbio, A1902), 3-Methyladenine (3-MA, Apexbio A8353R), Rapamycin (Rap, Apexbio, A8167), MK2206 (MK, Beyotime, SF2712). The antibodies used in our experiments were: anti-CDK1 antibody (Proteintech, 19532-1-AP), anti-Cyclin B1 antibody (Proteintech, 55004-1-AP), anti-Bax antibody (Proteintech, 50599-2-lg), anti- Bcl-2 antibody (Proteintech, 12789-1-AP), anti-Caspase-3 antibody (Proteintech, 19677-1-AP), anti-cleaved-caspase-3 antibody (CST, 9661S), anti-LC3 antibody (CST, 12741S), anti-Beclin 1 antibody (Proteintech, 11306-1-AP), anti-p62 antibody (Proteintech, 18420-1-AP), anti-AKT antibody (Proteintech, 60203-2-Ig), anti-Phospho-Akt (Ser473) antibody (CST, 4060S), anti-mTOR antibody (CST, 28273-1-AP), anti-Phospho-mTOR antibody (CST, 5536T), anti-β-Actin antibody (Proteintech, HRP-60008).

Cells were plated on coverslips, fixed with 4% paraformaldehyde for 1 h, washed three times with PBS and permeabilized with 0.5% Triton X-100 for 20 min. They were then treated in PBS with 2% bovine serum albumin (BSA) for 1 h and incubated overnight at 4 °C with anti-Caspase-3 antibody (1:100; Proteintech, Chicago, IL, USA, 66470-1-lg) or anti-Beclin1 antibody (1:100; Proteintech, Chicago, IL, USA, 11306-1-ap) diluted in 2% BSA. Finally, the cells were washed with PBS and incubated with a FITC-conjugated secondary antibody for 1 h in the dark. After washing, the cells were stained in PBS with DAPI. Subsequently, they were observed under a fluorescent microscope (Leica DMI8, Wetzlar, Germany).