Immunofluorescence triple staining

We stained a subset of sections for BrdU, Iba1 (ionized calcium-binding adaptor molecule), and glial fibrillary acidic protein (GFAP) to allow for BrdU positive cells to be phenotyped. Iba1 is expressed by macrophages and microglia, while GFAP is a type III intermediate filament protein expressed by astrocytes. This provides a means to quantify the proportion of Brdu + cells that also stain positive for the microglia/macrophage and astrocyte antibodies. DNA denaturation followed the same protocol as DAB staining; however, because we used HRP conjugated antibodies, PBS was used instead of TBS. Following denaturation, sections were blocked in 3% bovine serum albumin (BSA) with 0.25% Triton X-100 in PBS for 1 h at room temperature. They were then incubated overnight in the primary antibodies (see Table ​Table1)1) at 4 °C. Following washes in PBS and blocking solution, sections were incubated in the biotinylated goat anti-sheep antibody for 2 h, then in fluorophore secondary antibodies for 2 h at room temperature. Finally, sections were rinsed and mounted in 60% 2,2′-thiodiethanol (TDE), which has a high refractive index, making it advantageous for high-resolution confocal microscopy.