Rabbit anti il 4 receptor

Total protein extracts were obtained from the ipsilateral hemisphere of the brain 3 day after cerebral ischemia by homogenization in a 1X Cell Lysis Buffer (included in the Ray Biotech kit) with Protease inhibitor cocktail (Sigma). Non-specific binding was blocked by pre-incubation of the nitrocellulose membrane in PBS containing 0.1% tween 20 (PBS-T) and 5% skimmed milk for 1 ​h. The nitrocellulose was then incubated overnight at 4 ​°C with antibodies against the targeted proteins as follows: 1:500 rabbit anti Iba1 (Wako), 1:500 rat anti Gal- 3 (Cell Signaling), 1:500 rabbit anti Ym1 (Stem Cell technologies), 1:250 rabbit anti IL-4 receptor (Santa Cruz), 1:500 mouse anti phospho-p65 (Cell Signaling) and 1:20 ​000 rabbit anti actin (Santa Cruz). Primary antibody was detected with HRP-conjugated anti-rabbit or anti-mouse antibody (1:2000–1:5000) and blots were developed using an enhanced chemiluminescence detection system (ECL kit; Thermo Fisher Scientific). The density of the specific bands was quantified with Image J software and normalized to β-actin as a housekeeping protein (Lalancette-Hebert et al., 2011 (link)).

To validate in vivo imaging data mice from each experimental group were euthanized by overdose of anesthetic. For the double-immunofluorescence analysis, mice were perfused with PBS (pH 7.5) followed by 40 ​mg/ml paraformaldehyde and incubated overnight in 200 ​mg/ml phosphate-buffered sucrose. The sections were blocked in 10% goat serum and then incubated overnight at room temperature using primary antibodies diluted in 5% goat serum/PBS1x+0.25% Triton X-100 (1:500 rabbit polyclonal anti-Iba1 (Wako), 1:500 anti-Gal-3 (ATCC), 1:250 rabbit anti IL-4 receptor (Santa Cruz), 1:500 rabbit anti CD11b (Serotech), 1:500 rabbit polyclonal anti-Ym1 (Stem Cell technologies) and 1:500 rabbit anti actin (Invitrogen). After washes in PBS, the sections were then incubated in corresponding fluorescent goat secondary antiserum for 2 ​h (1:500, Invitrogen), washed and mounted with Flouromount G (Thermofisher) (Rahimian et al., 2019b (link)). Alexa Fluor® 488 phalloidin (Thermo Fisher Scientific) was used to study microglia morphology (Leica CTR 500 microscope).