Chicken anti gfap

Co-localization of vasculature, microglia and astrocytes was accomplished by first immunolabeling the vasculature with DAB and an antibody to CD31 as previously described above. After DAB visualization, the sections were washed 3 × 15 min in 0.4 % Triton X-100 buffer (this buffer was used as the medium for the remaining incubations and washes). The sections were then incubated in 5 % goat serum for 20 min at RT; subsequently, sections were incubated in a cocktail of chicken anti GFAP (1:500; Thermofisher scientific, USA) and polyclonal Rabbit IBA1 (Wako, Japan) for 48 h at 4 °C. After 48 h of incubation, sections were incubated in biotinylated goat anti chicken secondary IgG (1:200; Novex, USA) at RT for 2 h. Sections were then washed in PBS and Triton X-100 for 2–3 times. Then, sections were incubated in a cocktail of FITC-streptavidin (1:250; Vector Laboratories; CA, USA) and goat anti Rabbit Cy5 secondary IgG (1:150; Vector Laboratories; CA, USA). In this immunolabeling method green fluorescence denotes GFAP containing astrocytes and red fluorescence denotes IBA1 containing microglia.