Goat anti rabbit igg antibody

IVC tissues containing thrombus were fixed on slides and rinsed with PBS. The slides were blocked in 5% BSA in PBS for 1 h and treated with the primary antibodies at 4°C overnight, namely, rabbit anti-MLKL (p-S345) antibody (1 : 1000; #ab196436; Abcam, Cambridge, United Kingdom), rabbit anti-RIP3 (p-S232) antibody (1 : 50; #ab195117; Abcam, Cambridge, United Kingdom), and rabbit anti-IL-17B antibody (1 : 100; #ab79056; Abcam, Cambridge, United Kingdom). The slides were incubated at 37°C for 40 min with the secondary antibody (goat anti-rabbit IgG antibody, Abcam, Cambridge, United Kingdom) after washing with PBS three times and stained with hematoxylin for 5 min. The images were revealed using HRP-DAB and observed using a Lab.A1 microscope at 200x (Carl Zeiss GmbH, Oberkochen, Germany). The positive expression of the protein was observed by an optical microscope and showed brownish-yellow particles. Five high-power fields were randomly selected for observation using the double-blind method. The Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) was used to calculate the positive rate.

Proteins were extracted after cells were exposed to cisplatin (IC₅₀) for 48 h using ice-cold RIPA buffer (Beyotime, Chongqing, China) supplemented with phenylmethanesulfonyl fluoride (PMSF); the concentration was determined with a BCA kit (Beyotime). Proteins were separated by SDS-PAGE and transferred to a PVDF membrane (Merck Millipore, Billerica, MA, United States). Primary antibodies were as follows: anti-NTNG1 (GeneTex), anti-RAD51 (Abcam, Cambridge, United Kingdom), anti-AXL/p-AXL (Cell Signaling Technology, Danvers, MA, United States), anti-Akt/p-Akt (Cell Signaling Technol.), anti-GAS6 (Bioss Biotechnology, Beijing, China), and anti-β-actin (Proteintech, Wuhan, China). The secondary antibody was a goat anti-rabbit IgG antibody (Abcam). Bands were analyzed with the software Image Lab (Bio-Rad Lab., Hercules, CA, United States). The density ratio was used to calibrate the level of a target protein, with β-actin as the reference.
To detect the expression level of NTNG1 after cisplatin exposure, proteins were extracted after SKOV3 or SKOV3/DDP cells were exposed to cisplatin (IC₅₀ or 0.5 × IC₅₀) for 48 h, or after SKOV3/DDP cells were cultured in cisplatin-free medium for 3, 5, 7, and 9 days.

Liver cancer cells or tumor tissues of mice were prefixed in 4% paraform for 10 min, and fixed in pre-cold methanol for another 10 min. Next, cells were incubated with primary antibodies overnight at 4(C: anti-Ki67 (Abcam; 1:1000), anti-Smad4 (Abcam; 1:1000). Goat anti-rabbit IgG antibody (Abcam; 1:5000) was used as the secondary antibody. The nuclei were stained with DAPI for 15 min at room temperature. The samples were visualized by fluorescence microscope (Olympus CX23, Tokyo, Japan) immediately.

After the electrophysiological analysis, the distal segments near the ends of the nerve conduits from each group were fixed in 4% paraformaldehyde solution for 12 h, soaked in 30% sucrose solution at 4°C for 24 h until the samples sank to the bottom of the container. Then the tissue samples were frozen and cut into 8 μm–10 μm sections with a freezing microtome. The obtained sections were transferred onto glass slides and incubated in the PBS containing 1% normal goat serum for 1 h at 24°C. After that, the sections were incubated overnight at 4°C with anti-neurofilament antibody (anti-NF200, 1:200 dilution; Abcam, United States) and anti-S100 antibody (1:200 dilution; Abcam, United States), followed by the incubation for 1 h at room temperature with secondary antibodies (goat anti-rabbit IgG antibody and goat anti-mouse IgG antibody at 1:1,000 dilution; Abcam, United States). Images were acquired by a laser confocal microscope (FV1200; Olympus, Japan).