Pe rat anti mouse cd11b

Lung cells were suspended in a FACS buffer of PBS with 2 mmol/L EDTA (Thermo Fisher scientific) and 2v/v% FBS (Thermo Fisher scientific) and incubated with a cocktail of monoclonal antibodies, which included PE Rat Anti-Mouse CD11b, Anti-Mouse CD326 (EpCAM) APC (BD biosciences), Anti-Mouse CD45 FITC, Anti-Mouse F4/80 Antigen APC (ebioscience, San Diego, CA, USA), and PE/Cy7 Anti-mouse CD11c Antibody (Biolegend, San Diego, CA, USA). Flow cytometry was performed using a FACS Verse (BD Biosciences) and the FlowJo software program (Treestar). Alveolar macrophages were characterized as CD45+ CD11b- CD11c+ F4/80+, and lung epithelial cells were characterized as CD45- CD326+.

Purified anti-mouse CD16/32 antibody (Cat. no: 101312, Lot B298973), Alexa Fluor 488 conjugated anti-mouse Ly6G Ab (Cat. no: 127626, Lot B287100), PE conjugated anti-mouse Ly6G Ab (Cat. no: 127608, Lot B290416), Alexa Fluor 488 conjugated Rat IgG2a, κ Isotype control antibody (Cat. no: 400525, Lot B228070), PE conjugated Rat IgG2b, κ Isotype control antibody (Cat. no: 400608, Lot B170512), PE conjugated Mouse IgG1, κ Isotype control antibody (Cat. no: 400139, Lot B244868), PerCP/Cyanine5.5 Rat IgG2a, κ Isotype control antibody (Cat. no: 400531, Lot B268003) were purchased from Biolegend (San Diego, CA). PE conjugated anti-mouse ICAM-1(G-5) Ab (Cat. no: sc-8439; Santa Cruz Biotechnology, Santa Cruz, CA). Anti-VE-cadherin (F-8) (Cat. no: sc-9989 Lot J0217) (Santa Cruz Biotechnology, Santa Cruz, CA). Anti-VE-cadherin (D87F2) (Cat. no: 2500) (Cell Signaling Technology, Danvers, MA). PE Rat anti-mouse CD11b (Cat. no: 557397, Lot 7198506, BD Biosciences, San Jose, CA). APC-conjugated anti-mouse JAM-C(CD323) Rat IgG2b (Cat. no: FAB7050A, Lot ACHX0220011, R&D Systems, Minneapolis, MN). Alexa Fluor 568 goat anti-mouse IgG(H + L) (Cat. no: A11031, Lot 2216598, Invitrogen, Carlsbad, CA).

Mice were anesthetized with Euthasol (Virbac) and perfused with pre-perfusion buffer containing 0.5% sodium nitrite, 0.9% sodium chloride, and 0.1% heparin. The hippocampus was extracted and dissociated using the Neural Tissue Dissociation Kit (Miltenyi Biotec), according to manufacturer’s instructions. Microglia were isolated according to manufacturer’s instructions using Myelin Removal Beads II (Miltenyi Biotec). Microglia used for qRT-PCR analysis were further sorted using CD11b MicroBeads (Miltenyi Biotec) and immediately lysed for RNA extraction. Microglia used for single-cell RNA-seq analysis were FACS sorted using Live/Dead Fixable Violet (Life Technologies L34964), FITC rat anti-mouse CD45 (Clone 30-F11, BD Biosciences), and PE rat anti-mouse CD11b (Clone M1/70, BD Biosciences).