Anti caspase3 19677 1 ap

The frozen tissue sections or cells were allowed to recover to room temperature and then fixed with 4% PFA for 15 min, followed by permeabilization with 0.3% Triton X-100 for 15 min. Afterward, nonspecific antigen was blocked with 3% BSA for 1 h. Primary antibodies were added to the tissue and incubated at 4 °C overnight. On the following day, fluorescence-labeled secondary antibodies (Alexa Fluor 488 or tetramethylrhodamine conjugated, ZSGB-BIO, Beijing, China) were applied for 1 h at 37 °C in the dark. Finally, DAPI dye was used for nuclear counterstaining. All the primary antibodies were as follows: anti-Ki67 (28074-1-AP, Proteintech, Wuhan, China), anti-CASPASE3 (19677-1-AP, Proteintech, Wuhan, China), anti-P-MAPK (#4511, Cell Signaling Technology, Boston, USA), anti-KISS-1 (sc-18134, Santa Cruz, USA), anti-TER119 (sc-19592, Santa Cruz, USA), anti-SREBP2 (ab30682, Abcam, Cambridge, MA, UK), and anti-CD9 (#98327 S, Cell Signaling Technology, Boston, USA). All images were captured on a Leica confocal microscope (Weztlar, Germany).

Total protein was extracted using RIPA lysis buffer (Beyotime, China) and the concentration was detected using BCA Protein Assay Kit (Beyotime, China), then protein was separated in 10% SDS-PAGE and transferred to PVDF membrane (Millipore, Germany), after incubation with primary and secondary antibodies, every band was exposed in Chemiluminescene Imager (Tanon, China) using Ultra-sensitive ECL Chemiluminescene Kit (NCM Biotech, China). The antibodies used are as follows: anti-LRRTM4 (A15898, Abclonal), anti-caspase3 (19677-1-AP, Proteintech), anti-caspase9 (10380-1-AP, Proteintech), anti-Cyclin D1 (26939-1-AP, Proteintech), anti-Cyclin E2 (11935-1-AP, Proteintech), anti-E-cadherin (20874-1-AP, Proteintech), anti-N-cadherin (22018-1-AP, Proteintech), anti-SNAL1 (13099-1-AP, Proteintech), anti-GAPDH (60004-1-Ig, Proteintech).