Rabbit anti iba1

Coronal sections of 12 μm thickness were prepared adopting the standard protocol with minor modifications [34 (link)]. Briefly, sections were washed (PBS with 0.1% Triton X‐100), permeabilized (PBS with 1.5% Triton X‐100), blocked (PBS with 5% BSA) and incubated with the following primary antibodies: rabbit anti‐Iba1 (1 : 1000; Biocare Medical, Antwerpen, Belgium), rat anti‐MHC‐II (1 : 100; Abcam, Cambridge, UK), rat anti‐CD74 FITC (1 : 50; eBioscience) and mouse anti‐IRF1 (1 : 100; Santa Cruz Biotechnology, Heidelberg, Germany). Secondary antibodies against the appropriate species were incubated for 2 h at room temperature. Cell nuclei were counterstained with Hoechst (1 mg·mL⁻¹; Sigma). Sections were mounted on glass slides cover slipped using FluoromountTM Aqueous Mounting Medium (Sigma). For each brain section, at least 5 random 40× and 63× confocal images along the tumour margin and the tumour core were acquired with a Zeiss LSM880 microscope (Jena, Germany). High‐resolution XYZ stack images (1.024 × 1.024 pixels per Z step) were taken with a step size of 0.50 μm. Cell quantifications were performed using NIH imagej software (NIH, Bethesda, MD, USA) and values for single mouse are represented with distinct shape. Hoechst staining was used as reference for tumour localization.

Cells were cultured on poly-L-lysine coated coverslips and fixed with paraformaldehyde (4% in PBS) after stimulation. Cells were incubated overnight at 4°C with Cy3-labeled mouse anti-GFAP (1:800, Sigma-Aldrich), rabbit anti-Iba1 (1:200, Biocare Medical), goat anti-IL-1β (1:100, R&D), mouse anti-NLRP3 (1:100), rabbit anti-ASC (1:200), mouse anti-Caspase-1 (1:300, all three Adipogen) and rabbit anti-HMGB1 (1:400, Abcam). After washing with PBS, cells were incubated with anti-rabbit, anti-goat or anti-mouse secondary antibodies coupled to either Cy3 or AlexaFluor 488 (Invitrogen). Cells were then washed with PBS and mounted with DAPI-Fluoromount G (SouthernBiotech, USA), and observed under a LSM 510 META inverted confocal microscope (Carl Zeiss Micro Imaging, Göttingen, Germany).

Microglial cells were plated on poly-L-lysine coated coverslips. After treatments, cells were then fixed with paraformaldehyde (4% in PBS) for 20 min at room temperature, and permeabilized for 5 min in PBS containing 0.3% Triton X100. Following 3 washings in PBS, the blocking step was realized with 3% BSA in PBS at room temperature for 30 min. The cells were then incubated overnight at 4°C with a rabbit anti-Iba1 (1:300, Biocare Medical, USA). After washing steps with PBS, cells were incubated with Alexa488-conjugated anti-rabbit (1:1000, Life Technologies, Belgium) at room temperature for one hour. The cells were washed and mounted with Dapi-Fluoromount G (SouthernBiotech, USA). Cells were observed under a LSM 510 META inverted confocal microscope (Carl Zeiss Micro Imaging, Göttingen, Germany) at a 40-fold magnification.