Nf κb p p65 antibody

As for Western blot, the whole proteins were extracted with lysate buffer (RIPA buffer, Beyotime). Quantification was accomplished by the BCA protein assay kit (Beyotime). After that, the proteins were electrophoresed in a 10% SDS-PAGE gels electrophoresis for separation and transferred onto the polyvinylidene fluoride membrane. Blocking buffer was prepared with 5% bovine serum albumin (Biosharp, Anhui, China). Then, they were immunoblotted with the primary antibodies (1:1,000) and detected with horseradish peroxidase-conjugated goat anti-rabbit-IgG (1:10,000, Proteintech, Wuhan, China). Then, the membranes were visualized by using Gel-Pro-Analyzer Software (Beijing, China). The primary antibodies involved in this procedure are listed as follows: LAPTM5 antibody, MCP1 antibody, and CD86 antibody were obtained from ABclonal (China). nuclear factor-kappa B (NF-κB) p65 antibody, NF-κB p-p65 antibody, p-IκBα antibody, and IκBα antibody were purchased from Affinity (Jiangsu, China).

Immunofluorescence (IF) staining was constructed by the method described in many studies. The tissue was embedded in paraffin and cut into 5-µm sections beforehand. All slides were deparaffinized, rehydrated in xylene, 100%, 95%, 85%, 75% ethanol, and PBS, and constantly stained with 10% antigen retrieval solution for 10 min. BSA (1%, Sangon) as the blocking buffer was incubated at room temperature for 15 min. The primary antibody was added and incubated at 4°C overnight. Then, the secondary antibody, and 4′,6-diamidino-2-phenylindole (DAPI) (Aladdin) were added to this procedure. The antibodies used in this section involved as follows: NF-κB p-p65 antibody (1:200, Affinity, Jiangsu, China), CD68 (1:50, Santa Cruz, Santa Cruz, CA, USA), iNOS (1:100, ABclonal), Cy3 labeled goat anti-mouse IgG (1:200, Invitrogen, Carlsbad, CA, USA), Cy3 labeled goat anti-rabbit IgG (1:200, Invitrogen), FITC labeled goat anti-mouse IgG (1:200, Abcam, Cambridge, UK). The immunofluorescence images were taken and preserved using an OLYMPUS-BX53 microscope (OLYMPUS, Tokyo, Japan).