Pacific blue conjugated anti cd45

The following mouse-specific IgG monoclonal antibodies were purchased from BioLegend (San Diego): biotin-conjugated anti-MHCII; Brilliant Violet 605-conjugated streptavidin; Pacific Blue–conjugated anti-CD45; allophycocyanin-conjugated anti-F4/80; fluorescein isothiocyanate-conjugated anti-B220; phycoerythrin-conjugated anti-IL-10R and anti-SH; PerCP-Cy5.5-conjugated anti-CD11b; PE-Cy7-conjugated anti-CD11c; and Brilliant Violet 510-conjugated anti-CD103. Fixable Viability Dye eFluor780 was purchased from eBioscience (San Diego). Single-cell suspensions from primary tissues were stained with a suitable combination of fluorochrome-conjugated antibodies and Fixable Viability Dye, fixed in 2% paraformaldehyde in PBS, and examined with a BD LSR II flow cytometer (Becton, Dickinson). The data were analyzed with FlowJo software. For counting of cells by flow cytometry, Flow-Count Fluorospheres (Beckman Coulter, Brea, CA) were used per instructions provided by the manufacturer.

Central nervous system tissues including brain and spinal cord were collected and macerated with cold PBS in small petri dishes that were kept cold (on ice). Cell suspensions were strained with a 70-µm strainer and mononuclear cells are isolated with 30% percoll gradient. The pelleted cells were washed and pre-blocked for Fc receptors using anti-CD16/32 (Fc-block). Cells were then incubated with monoclonal antibody at 4°C for 30 min, after which cells were washed with PBS containing 2% FBS. For flow cytometry antibodies the CD16/32 Fc-block, PE conjugated anti-F4/80, Pacific Blue conjugated anti-CD45, APC-Cy7 conjugated anti-Ly6C and/or Alexa 488 conjugated anti-CD206 were all obtained from Biolegend (San Diego, CA, USA). Anti-mouse Ron was purchased from R&D Systems (Minneapolis, MN, USA), and the secondary antibody was Alexa 488 conjugated to anti-goat for Ron. Stained cells were analyzed on a BD LSR Fortessa (BD Biosciences), and the flow output was analyzed with FlowJo software (Tree Star).

P12 mice were anesthetized by intraperitoneal administration of a cocktail of domitor (0.3 mg/kg), dormicum (4 mg/kg), and butorphanol (5 mg/kg). Mice were transcardially perfused with ice-cold PBS, and dissected brains were digested with collagenase D (1 mg/mL; Roche) containing 2.5 mM calcium chloride at 37 °C for 30 min. After trituration, digested tissues were resuspended with 30% percoll (GE Healthcare), and 70% percoll was layered underneath, followed by centrifugation at 2000 rpm for 30 min at room temperature. Cells were treated with APC conjugated anti-CD11b (1:200; Biolegend) and pacific blue conjugated anti-CD45 (1:200; Biolegend) on ice for 30 min. GFP⁺ CD11b⁺ CD45^mid microglia were sorted with cell sorter SH800 (Sony). Total RNA was purified from sorted microglia by Trizol Reagent (Thermo Fisher Scientific). Quantitative RT-PCR was conducted with Quant-X One-Step qRT-PCR TB green Kit (Takara) and CFX Connect Real-time PCR Detection System (Bio-rad). Sequences of PCR primers of Igf1 are as follows: 5′-gtgagccaaagacacaccca-3′ (forward) and 5′-acctctgattttccgagttgc-3′ (reverse).