Anti cd86

Brain tissues from mice were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 4 µm-thick segments. After heat-induced antigen epitope retrieval, slides were blocked with PBS containing 3% normal serum and incubated overnight at 4 °C with primary antibodies as follows: anti-CD68, anti-CD86 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-CD4, anti-CD8, anti-Iba-1, and anti-TSPO (Abcam, Cambridge, UK). The slides were then washed with PBS and incubated with biotinylated secondary antibodies (anti-goat for CD68; anti-rabbit for CD4, CD8, and Iba-1; all from Santa Cruz Biotechnology). The slides were then treated with avidin-biotin solution (Vector Laboratories, Burlingame, CA, USA) for 1 h. The antigenic signal was developed using DAB (3, 3 -diaminobenzidine) substrate (Vector Laboratories, Burlingame, CA, USA), according to the manufacturer's instructions. Finally, the slides were counterstained with hematoxylin and mounted with the Permount Mounting Medium (Thermo Fisher Scientific, Fair Lawn, NJ, USA).

Clarified cell lysates separated by 10% SDS-PAGE were transferred to PVDF membranes and probed with an anti-arginase 1 (Arg1) antibody, developed in mice (cat. #SC-21738, Santa Cruz Biotechnology, Dallas, TX, USA), anti-CD86 (cat. #ab53004, Abcam, Boston, MA, USA), and anti-Iba1 (cat. #016-20001, Wako Chemicals USA Inc., Richmond, VA, USA), developed in rabbits, diluted 1:1000, and followed by addition of the corresponding secondary antibodies (cat. #A9169, Sigma-Aldrich, Saint Louis, MO, USA). Detection was performed with enhanced chemiluminescence methodology (cat. #34075, SuperSignal^® West Dura Extended Duration Substrate; Pierce, Rockford, IL, USA), and the intensity of the signal was measured using a gel documentation system (Versa Doc Model 1000, Bio-Rad, Hercules, CA, USA). β-tubulin (cat. #86298S, Cell Signaling, Danvers, MA, USA) immunoreactive signal was used as the loading control.

For histological analysis, the left lung tissue was fixed with 4% paraformaldehyde for H&E or immunohistochemical (IHC) staining after paraffin embedding. Briefly, 4-μm tissue sections were deparaffinized using EZ prep (Ventana Medical Systems). The slides were incubated with anti-F4/80 (Santa Cruz, sc-377009), anti-CD86 (Santa Cruz, sc-28347), and anti-CD206 (Santa Cruz, sc-376108) using the automated Ventana Benchmark XT (Ventana Medical Systems), and detected using the Ultra view DAB Detection Kit (Ventana Medical Systems) following the manufacturer’s protocol. A microscope (OLYMPUS, BX51) was used to randomly select regions at 200× and 400× magnifications. Staining intensity was quantified using ImageJ software with the IHC toolbox plugin. Lung injury scores were assessed following the guidelines outlined in the American Thoracic Society Workshop Report.⁴³ (link)