Goat anti rabbit igg h l hrp

RIPA lysis buffer containing protease inhibitor (Beyotime Biotechnology, Shanghai, China) was used to lyse the GC cells at 4°C, and centrifuged at 12,000 r/min for 5 min, with the supernatant collected as the whole protein extract. The BCA protein quantification kit (Beyotime Biotechnology, Shanghai, China) was employed to determine the protein concentration. Next, the protein samples were mixed with loading buffer, and the proteins were denatured in boiling water for 5 min. The protein samples were dissolved via sodium dodecyl sulfate polyacrylamide gel electrophoresis, then transferred to polyvinylidene fluoride (PVDF) membrane, which was then blocked by 5% skimmed milk for 2 h at ambient temperature. Rabbit anti-CXCL12 antibody (cell signaling technology, 3530, 1:1000) and rabbit anti-β-actin antibody (Abcam, ab8227, 1:1000) were loaded and the membrane was incubated overnight at 4°C. Next, the membrane was washed with tris buffered saline tween (TBST), and then incubated with goat anti-rabbit IgG H&L (HRP) (Abcam, ab205718,1:2000) for 1 h. After the PVDF membrane was washed in TBST, the protein bands were developed by a high-sensitivity ECL chemiluminescence kit (Beyotime, Shanghai, China).

Tissue microarray (TMA) slides were subjected to the following procedures: first, dewaxing with xylene, followed by rehydration through a graded ethanol series. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide (Sinopharm Chemical Reagent Co., China, #73113760) for 10 min. The slides were then rinsed in PBS, underwent hot antigen repairing, and were subsequently washed with PBS. Next, 5% BSA (Sigma, Shanghai, China, # B2064) was added to each slide and incubated at room temperature for 20 min. Primary antibodies were then added to the slides in a volume of 100 μL each: COQ2 (Sino Biological, Rabbit anti-human mAb, 1:100, 206810-T08), MPC1 (SAB, Rabbit anti-human mAb, 1:200, #42898), and ADAMTS13 (SAB, Rabbit anti-human mAb, 1:150, #49953). The slides were incubated overnight at 4°C. The following day, the slides were rinsed with PBS and incubated with a labeled secondary antibody (Abcam, Goat anti-rabbit IgG H&L (HRP), 1:2000, ab205718) at 37°C for 30 min. After rinsing again with PBS, each section was treated with 100ul try-488 Tyramine Conversion Reagent (runnerbio, Bry-try488) and incubated for 10–30 min at room temperature. Finally, the sections were mounted with anti-fluorescence quenching sealer containing DAPI (Beyotime Biotechnology, China, P0131).

Total protein was extracted from each sample, separated on 10% SDS-PAGE, and transferred to PVDF membranes. The membranes were then blocked with 5% skimmed milk powder in TBST for 2 h and immunoblotted with anti-B7–H3 (1:500, GTX34211, GeneTex, Inc., Texas, USA) and anti-β-actin (1:1000, GTX109639, GeneTex, Inc.) at 4 °C overnight. Afterward, the membrane was incubated with goat anti-rabbit IgG H&L (HRP) (Abcam, Cambridge, UK, ab6721, 1:2000) at 37 °C for 2 h. The proteins were visualized using enhanced chemiluminescence reagent (Thermo, USA), and the images were processed using Quantity One 4.4.0 software.

Initially, a radio-immunoprecipitation assay (RIPA) lysis buffering solution (Beyotime, Shanghai, China) was applied to abstract proteins. Following the separation of proteins by sodium salt-polyacrylamide gel electrophoresis, target proteins were electrophoretically moved to nitrocellulose films, blocked via 5% milk-TBST for 2 h under room temperature. Subsequently, they were washed three times in TBST buffering solution and cultivated with the first anti-substances, viz., COL5A1, COL1A1, E-cadherin, N-cadherin, snail, GADPH rabbit polyclonal antibody (Proteintech, USA), vimentin, and vinculin mouse polyclonal antibody (Proteintech, USA) at 4°C overnight. Subsequently, the films were washed three times in TBST again, each for 10 min. Goat anti-rabbit IgG H&L (HRP) and goat anti-mouse IgG H&L (HRP) (Abcam, USA) were added then for cultivation. Afterwards, the membranes were cleaned in TBST as per the steps mentioned above to analyze relative protein expression with Image-Pro Plus software 6.0.

Total proteins from brain tissues with the hippocampus were extracted with a Total Protein Extraction Kit (BC3711, Solarbio) and quantified by the BCA protein quantification kit (ab102536, Abcam, Cambridge, UK) in line with the operation manual. 10 %SDS-PAGE was used to dissolve protein samples and electrically transfer them to PVDF membranes. For the block, the membrane was treated for 1 h at room temperature with 3% bovine serum albumin (BSA) and then primed overnight at 4 °C with primary antibodies. As a next step, the membrane was washed three times and hatched for 1 hour at 37 °C with appropriate secondary antibodies. An ECL chemiluminescence kit (WBULS0500; EMD Millipore) was used to visualize the bands. The primary antibodies were NLRP3 (1:1000), Iba-1 (1:10,000), IL-18 (1:500), IL-1β (1:1000), and caspase1 (1:1000), and the second antibody was Goat anti-rabbit IgG H&L (HRP) (1:20,000), all of which were purchased from Abcam.

Vanillic acid was purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). The primers were supplied by Sangon Biotech (Shanghai, China). The enzyme-linked immunosorbent assay (ELISA) kits for IL-1β and IL-18 were supplied by Invitrogen (Life Technologies Corp. California, United States). Fetal bovine serum (FBS), bovine serum albumin (BSA), Dulbecco’s Modified Eagle’s Medium (DMEM), TRIzol, and 0.25% trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA) were purchased from Gibco (Life Technologies Corp., California, United States). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Kumamoto, Japan). Antibodies against NLRP3, ASC, caspase-1, CGRP, NGF, TrkA, and type I collagen were purchased from Abcam (Cambridge, United Kingdom). Monoidoacetate acid, type I collagenase, and dimethylsulfoxide (DMSO) were all obtained from Sigma (St Louis, USA). All other chemicals were of reagent grade. Goat anti-rabbit IgG H&L (HRP) and Picro Sirius Red Stain kit were also supplied by Abcam (Cambridge, United Kingdom).