Alexa fluor 488 conjugated anti mouse and alexa 546 conjugated anti rabbit antibodies

PFA-fixed brain sections containing infarct core and penumbra were blocked with 5% bovine serum albumin in Tris-buffered saline with 0.5% v/v Triton X-100 (TBS-T) and incubated with anti-Iba-1 (1:200; Wako, Richmond, VA, United States) and anti-arginase-1 antibodies (1:200; Invitrogen) for 48 h. Sections were washed three times with TBS followed by 2 h incubation at RT with Alexa Fluor 488-conjugated anti-mouse and Alexa 546-conjugated anti-rabbit antibodies (1:2000 each; Invitrogen, Carlsbad, CA, United States) in TBS. Images were obtained in a Zeiss LSM 800 confocal microscope using a 63X objective. An average of 45 optical slices was obtained every 0.5 μm for each Z-stack.

Fixed spinal tissues were cut into 40 μm thick transverse sections with a cryostat. Sections adjacent to the site of the lesion were blocked with 5% bovine serum albumin in Tris-buffered saline with 0.1% v/v Triton X-100 (TBS-T) and incubated with anti-AQP4 (1 : 50; Genetex, Irvine, CA) and anti-GFAP antibodies (1 : 2000; Sigma, St. Louis, MO) for 24 h. Sections were washed 3 times with TBS followed by 2 h incubation at room temperature with Alexa Fluor 488-conjugated anti-mouse and Alexa 546-conjugated anti-rabbit antibodies (1 : 2000 each; Invitrogen, Carlsbad, CA) in TBS. Images were obtained by confocal microscopy (Leica TCS SP5) using a 63x objective. An average of 60 optical slices was obtained every 0.2 μm for each Z-stack. Fluorescence intensity quantification was performed with the image processing package Fiji for ImageJ (NIH) analyzing 8-bit grayscale photograms of images calibrated by area; four images per region were analyzed and averaged for each group. Quantitative data is expressed as arbitrary units of fluorescence.