Goat anti rabbit igg secondary antibody

LLC CAFs were obtained from the s.c. implanted LLC tumor by collagenase treatment, following separation with meshing by a nylon filter and adhesion of plastics, as previously described (1 (link),2 (link)). CAFs were incubated in DMEM media supplemented with 20% FBS at 37°C and passaged every 3–4 days. After 5 passages, cells were fixed with 4% paraformaldehyde (BioSharp Life Sciences) for 15 min, and subsequently permeabilized with 0.2% Triton-X-100 (Beijing Dingguo Changsheng Biotechnology Co., Ltd.) for 15 min at room temperature. Cell were blocked with 10% goat serum (Biological Industries) at room temperature for 1 h and incubated with the following primary antibodies: Rabbit anti-α-smooth muscle actin (1:500; cat. no. NBP1-30894; Novus Biological, Ltd.), anti-FAP-α (1:500; cat. no. MAB9727; Novus Biological, Ltd.), anti-vimentin (1:500; cat. no. NBP1-97670; Novus Biological) or anti-pan cytokeratin (1:200; cat. no. NB600-579; Novus Biological) overnight at 4°C. Cells were washed three times with PBS and subsequently incubated with a goat anti-rabbit IgG secondary antibody (1:500; cat no. SA00001-2; ProteinTech Group, Inc.) for 1 h at 37°C. The nuclei of the CAFs were stained with DAPI (ProteinTech Group, Inc.) and cell images were captured using an OLYMPUS IX73 fluorescence microscope (magnification × 400; Olympus Corporation).

Total protein was extracted from cells by washing them with pre-cooled PBS and lysing with 200 µL 1 × loading buffer. Proteins were then run on sodium dodecyl sulfate–pulsed field gel electrophoresis, and electroblotted onto polyvinylidene fluoride membranes. Membranes were incubated with a primary anti-GnRHR antibody (Immunoway, Suzhou, China) at 4°C overnight, then incubated with a goat anti-rabbit IgG secondary antibody (Proteintech Group, Wuhan, China) for 1 hour at room temperature. for 1 hour at room temperature. Visualization was carried out using a chemiluminescence system (Leica).