Anti rabbit antibodies

apoc1 protein expression was detected in GC and adjacent tissues by IHC. Formalin-fixed paraffin-embedded tissues sections were degreased in xylene and rehydrated in different concentrations of alcohol and distilled water before antigen retrieval, then followed by antigen retrieval. The fixed sections were washed three times in phosphate buffered saline (PBS) (pH 7.3), incubated in 3% H₂O₂ for 7 min, and then washed three times with PBS again. The sections were blocked with BSA (Servicebio, Wuhan, China) for 30 min, followed by incubation with Anti-apoc1 primary antibody (Abcam, USA) at 4°C overnight, followed by incubation with anti-rabbit antibodies (Proteintech Technology, Wuhan, China) for 50 min. Subsequently, the sections were incubated with diaminobenzidine as a chromogen for 5 min. The sections were then counterstained with hematoxylin and dehydrated using ethanol and xylene. The sections were viewed under an inverted microscope (Nikon Corporation, Japan).

The steps are based on the procedures used by Liu et al. [21 (link)]. In brief, in keeping with the manufacturer’s protocol, total protein concentration was detected using the BCA kit obtained from the Beyotime Institute of Biotechnology (Shanghai, China). Samples in the ileal mucosa were obtained via SDS-PAGE and transferred to PVDF membranes (Millipore, Eschborn, Germany). The membrane was blocked and then incubated with primary antibodies overnight (4 °C). The membrane was rinsed and then incubated with the secondary antibodies for 60 min (25 °C). The antibodies, including glucose-regulated protein-78 (GRP78), activating transcription factor 6 (ATF6), gasdermin-D (GSDMD), caspase-1, caspase 3, and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) conjugated anti-rabbit antibodies were supplied by Proteintech Group, Inc. (Wuhan, China). The signal was visualized by extreme hypersensitivity ECL chemiluminescence kit (Beyotime, Shanghai, China), and the value was displayed using the Image lab analysis software (version 6.1, Bio-Rad. Berkeley, CA, USA).

Cells at a concentration of 1 × 10⁵ cells/mL were plated into a 6-well dish. After 3 days of incubation, cells in each well were lysed with lysis buffer at 4 °C for 30 min. The lysates were loaded and analyzed using SDS-PAGE following standard protocols. Primary antibodies used were anti-REG4 antibody (1:500, Abcam, Cambridge, UK) and anti-Actin antibody (1:1000, Proteintech, Wuhan, China). Secondary antibodies used were anti-mouse antibodies (1:1000, Proteintech, Wuhan, China) and anti-rabbit antibodies (1:1000, Proteintech, Wuhan, China).