Immunofluorescence Analysis of FRT Tissue

FRT tissues were embedded in Tissue-Tek OCT (Sakura, Netherlands). Sections were fixed with acetone, and blocked (50 μg/ml mouse IgG and 10% BSA) and stained with: Ly6g 1A8 (Biolegend), F4/80 BM8 (Biolegend), MHCII 2G9 (BD Biosciences), CD31 2H8 (Invitrogen), α-SMA 1A4 (eBioscience), CD62-E (BD Bioscience), CD62P REA344 (Miltenyi Biotec), ICAM-1 M-19 (Santa Cruz Biotechnology), CD106 429 (Miltenyi Biotec). For protein expression quantification, tissues were imaged using the glycerol ACS APO 20x NA 0.60 objective of a confocal fluorescence microscope (SPE, Leica Microsystems), maintaining the acquisition settings all over the process for each sample and among samples, as previously described (19 (link)). Mean fluorescence intensities (MFI) were assessed at multiple regions of interest (ROIs) by field, using the glycerol ACS APO 63x objective and randomly depicted at specific areas of the venular beds in FRT. All quantifications were performed using the FIJI (Fiji Is Just ImageJ) software (NIH).