Anti-GFAP Antibody Picoband®

Boster bio GFAP 1:100 PFA (7-2) - cortex 12x - with scale bar

Boster bio GFAP 1:100 PFA (7-2) - cortex 20x - with scale bar

Boster bio GFAP 1:250 12x (free-floating)

Images that were made from fresh frozen cryostat sections, were thaw-mounted onto microscope slides. Mounted sections were later post-fixed with 300 µL of 4% paraformaldehyde (PFA) in PBS at room temperature for 10 minutes. After fixation and prior to incubation with antibodies, slides were washed 3 times with 300 µL of 1X PBS with 0.01% sodium azide for 3 minutes per rinse, followed by permeabilization with 300 µL of 0.3% Triton X-100 in 1X PBS with 0.01% sodium azide for 30 minutes at room temperature, then blocking with 700 µL of 4% donkey serum diluted in 1X PBS with 0.01% sodium azide + Triton X-100 (blocking buffer) for 30 minutes at room temperature. Slides were incubated with 325 µL of GFAP (1:100). Concentrations of 1:100 were achieved by diluting 10 µL of antibody in 1,000 µL of blocking buffer; concentrations of 1:250 were achieved by diluting 4 µL of antibody in 1,000 µL of blocking buffer. Sections were incubated overnight at 4 ˚C. On the next day, slides were washed 3 times with 300 µL of 1X PBS with 0.01% sodium azide for 3 minutes per rinse, then incubated with the secondary antibodies at room temperature, in the dark, for 1 hour. Washing step was repeated then slides were left in the dark to dry. Mounting media was added to cover slip the sections. Slides were kept in the dark at 4 ˚C prior to imaging.

Immunohistochemistry was also performed with free-floating sections, which were exposed to the same primary antibodies diluted (1:250) in blocking buffer overnight and, subsequently, exposed to secondary antibodies diluted in blocking buffer for 1 hour, following the same procedure as the mounted sections. After incubation, sections were washed 3 times with 300 µL of 1X PBS with 0.01% sodium azide for 3 minutes per rinse and transferred with a painting brush to a container filled with 1X PBS with 0.01% sodium azide, from which they were mounted onto slides. Slides were left to dry in the dark, after which mounting media was added to cover slip the sections. As with slide-mounted sections, these slides were kept in the dark at 4 ˚C prior to imaging.