Anti rabbit igg secondary antibodies

H&E staining was used to observe the neuronal morphology and damage, while immunohistochemistry was performed to detect the activation of NLRP3 and microglia in the rat hippocampus. The brain tissues were embedded in paraffin, and 5-μm thick brain sections were prepared using a slicer (RM2235, Leica, Germany). After dewaxed and rehydrated by xylene and ethanol, the slides were stained with H&E staining or immunohistochemistry. H&E staining was conducted on 5 μm sections using standard H&E staining protocol. For immunohistochemistry, the sections were subjected to induced antigen retrieval in citrate buffer at 100°C (0.01 M, pH = 6.0), and then immersed in 3% hydrogen peroxide solution for 10 min at RT to quench endogenous peroxidase activity. After blocking with 5% bull serum albumin, the tissues were incubated with anti-NLRP3 antibody (1:50, BA3677, Boster, Wuhan, China) or anti-Iba-1 antibody (1:500, ab178846, Abcam, USA) overnight at 4°C. Afterward, the sections were rinsed with PBS three times and incubated with anti-rabbit IgG secondary antibodies (1:200, Boster, Wuhan, China) at RT for 1 h, and then were viewed by 3,3′-diaminobenzidine solution and counterstained with Hematoxylin. The sections were visualized and analyzed by a light microscope (Olympus, Tokyo, Japan) equipped with an imaging system and Image J software.

Western blotting was used to detect indicators of osteogenic differentiation, such as collagen type I (COL 1), runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN). After 14, 21, and 28 days of osteogenic induction, the cells on the samples were collected by trypsinization. The cells were lysed using RIPA lysis buffer (Beyotime) and centrifuged to obtain cell lysates. Protein quantification of whole cell lysates was performed using the BCA protein assay kit (Beyotime). Aliquots of cell lysates at a concentration of 20 µg/µL were loaded onto SDS-PAGE systems and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with blocking solution (Beyotime) and incubated overnight with primary antibodies (1:1,000; Abcam, Cambridge, UK), followed by washing and incubations with anti-rabbit IgG secondary antibodies (Boster, Wuhan, People’s Republic of China). The primary antibodies used were anti-COL1, anti-OCN, and anti-RUNX2; anti-β-actin was used for housekeeping. Finally, the Western blot membranes were exposed to Luminata Forte Western horseradish peroxidase (HRP) substrate (EMD Millipore, Billerica, MA, USA) and analyzed using a chemiluminescence system. The immunoblot bands were analyzed quantitatively with ImageJ software.