Ab33827

Immunohistochemistry and immunocytochemistry were performed according to the routine methods. Dilutions of the primary antibodies were as follows: rat anti-MBP (1:200; SAB2108749, Sigma-Aldrich), rabbit anti-Olig2 (1:200; 387R-1, Sigma-Aldrich), rabbit anti-NG2 (1:200; SAB5700198, Sigma-Aldrich), mouse anti-NG2 (1:300, 14–6504-82, Sigma-Aldrich), monoclonal antibody CC1 (1:200, 17–0661-82, Thermofisher Scientific), rabbit anti-PDGFa (1:500, PA5-50,565, Thermofisher Scientific), mouse monoclonal anti-Ki67 antibody (1:200, MIB1, SANTA CRUZ), polyclonal anti-ADAM10 (1:100; ZRB2367; Sigma-Aldrich), anti-GFAP (1:200, MA5-12,023, Thermofisher Scientific), anti-NeuN (1:100, 66,836–1-Ig, Proteintech), and anti OX42 (1:300, ab33827, Abcam). Samples were incubated with corresponding primary antibodies overnight at 4 °C, followed by rinsing in PBS, and stained with Cy3 (1:1000; Invitrogen, Carlsbad, CA, USA) or Alexa Fluor 488-conjugated secondary antibodies (1:1000; Invitrogen) for 1 h at room.

Rats were deeply anesthetized before undergoing intracardiac perfusion with 4% paraformaldehyde. The SLNs were isolated and embedded in OCT compound (Sakura Finetek, Tokyo, Japan), and 20 μm sagittal sections were generated with a cryostat (Leica CM3050S, Leica Biosystems, Denver, CO). The sections were permeabilized with 0.1% Triton X-100 in phosphate buffered saline for 20 min, blocked with 5% bovine serum for 30 min and incubated overnight with the following primary antibodies: mouse anti-rat CD31 (1:40, 550300 BD Pharmingen), rabbit anti-CD206 (1:1000, ab64693, Abcam) and mouse anti-CD11b (1:1000, ab33827, Abcam). The following secondary antibodies were used: anti-mouse IgG Alexa Fluor 488 and anti-rat IgG Alexa Fluor 647. After counterstaining with 4',6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich), tissue images were observed through a universal fluorescence microscope (BZ9000; Keyence Co., Osaka, Japan).