Immunofluorescence Staining Protocol for Rat Brain

The procedure and protocol for IF staining have been reported in our previous studies [17 (link)–19 (link)]. For IF staining, rehydrated paraffin sections were first treated with 3% H₂O₂ for 30 min and incubated with Immuno-Block reagent (BioSB, Santa Barbara, CA, USA) for 30 min at room temperature. Sections were then incubated with primary antibodies specifically against CD31 (1:100, Bio-Rad), von Willebrand factor (vWF) (1:200, Merck Millipore), NeuN (1:1000, Millipore, Billerica, MA, USA), stromal cell-derived factor [(SDF)-1α (1:100, Santa Cruz Biotechnology), vascular endothelial growth factor (VEGF) (1:400, Abcam), CXCR4 (1:200, Thermo Fisher Scientific), microglial cell (1:100, Abcam, Cambridge, UK), CD68 (1:100,Abcam, Cambridge, UK), and glial fibrillary acidic protein (GFAP) (1:500, Dako) at 4 °C overnight. Alexa Fluor488, Alexa Fluor568, or Alexa Fluor594-conjugated goat anti-mouse or rabbit IgG were used to localize signals. Sections were finally counterstained with DAPI and observed with a fluorescent microscope equipped with epifluorescence (Olympus IX-40).
Three brain sections were analyzed for each rat. For quantification, three randomly selected high-power fields (HPFs; × 400 for IF study) were analyzed in each section. The mean number of positively stained cells per HPF for each animal was then determined by summation of all numbers divided by 9.