Cd68 antibody

Colonic tissues were fixed in 4% (w/v) paraformaldehyde overnight and embedded in paraffin. Five μm sections were then sent for dewaxing and rehydration. After the sections were blocked with 5% bovine serum albumin in PBS for 2 h, they were incubated with lysozyme antibody (1:250, Abcam, Cambridge, MA, USA), CD68 antibody (1:100, Proteintech Group Inc., Chicago, IL, USA), CD206 antibody (1:100,; Proteintech Group Inc., Chicago, IL, USA) or LC3 antibody (1:200, Novus Biologicals, Littleton, CO, USA) overnight at 4 °C. After washing three times with PBS, the sections were stained by Alexa-488- or Alexa-Cy3-labeled secondary antibody (1:500, Jackson ImmunoResearch Inc., West Grove, PA, USA) for 30 min at 37 °C. After washing, the slides were mounted with Vectashield mounting medium containing 4′,6-Diamidino-2-Phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA) and colocalization was observed using a confocal laser scanning microscope (Fluoview FV1000, Olympus, Tokyo, Japan). In this study, the experiments were performed in a double-blind manner.

Proteins were extracted from the colonic tissues or murine BMDM using a standard extraction reagent supplemented with the protease inhibitor (KANGCHEN; Shanghai, China). The protein concentration was determined using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Haimen, China). Proteins were then separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and electro-transferred to nitrocellulose membranes as described previously^{68 (link)}, and incubated with a primary antibody overnight at 4 °C. The samples were then incubated with an IRDye800CW-conjugated secondary antibody (Rockland, Gilbertsville, PA, USA) for 1 h at 25 °C. The image was acquired with the Odyssey infrared imaging system (Li-Cor Biosciences, Lincoln, NE, USA). All of the immunoblotting experiments were repeated at least five times. The following primary antibodies were used: Beclin-1 antibody (1:500; Cell Signaling Technology, Danvers, MA, USA), light chain 3 (LC3) antibody (1:500; Novus Biologicals, Littleton, CO, USA), and p62 antibody (1:500; Cell Signaling Technology, Danvers, MA, USA), CD68 antibody (1:300; Proteintech Group Inc., Chicago, IL, USA), and CD206 antibody (1:500; Proteintech Group Inc., Chicago, IL, USA).