Immunofluorescence staining for CD34, Gr1 and von Willebrand Factor

Immunofluorescence (IF) was performed to localize the neutrophil marker, Gr1 and endothelial and platelet marker, vWF (Von Willebrand Factor), in WT and CD34 KO mice across different time-points. Tissue sections were deparaffinized and rehydrated followed by inactivation of endogenous peroxidase activity with 0.5% H₂O₂ in methanol in dark for 20 min at room temperature. After washing, antigen retrieval was performed in two steps. Firstly, heat induced epitope retrieval was done by incubating sections in sodium citrate buffer (10 mM, pH 6.0) at 90–95 °C for 20 min. Then, the sections were allowed to cool in distilled water, following which the sections were incubated with warmed pepsin (2 mg/ml in 0.01 N HCl) at 37 °C for 20 min. After washing, the sections were blocked with 1% bovine serum albumin in PBS for 30 min at room temperature and then incubated overnight at 4 °C with 100 µL primary antibodies per section against rabbit vWF (1:300 dilution, Catalogue A0082, Dako Denmark A/S, Glostrup, Denmark), rat Gr-1 (1:100 dilution; Catalogue 550,291; BD Biosciences, ON, Canada), and the sections were incubated overnight at 4 °C with 100 µL primary antibodies per section against CD34 (rabbit anti-mouse, EP373Y, ab81289, 1:500, Abcam Inc., Cambridge, MA, USA). Next day, the slides were left at room temperature for 30 min followed by washing with PBS 1X thrice. After washing, they were incubated in dark with 100 µL secondary antibody per section (AF488, green) for 30 min at room temperature, counterstained with DAPI, and dried. The cover slips were mounted with Prolong gold mounting medium, sealed with nail varnish and stored in dark at 4 °C.