Lsm880 airyscan confocal microscope

Mice were anesthetized using isoflurane and transcardially perfused with ice-cold phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA) solution in PBS. Brains were dissected and stored overnight in 4% PFA and transferred to a 30% sucrose solution until tissue density reached equilibrium and brains sank to the bottom of their holding tubes. 40 μm brain sections were taken using a Leica CM3050 S cryostat (Leica Microsystem, Weitzlar, Germany). Brain sections were then washed in Tris-Buffered Saline (TBS) 3X for 10 min, followed by 30 min of block in TBS with 40% normal goat serum and 0.2% Triton X-100 (TBS+). Sections were then incubated in primary antibody diluted 1:500 in TBS + overnight at room temperature with gentle agitation. The next day, sections were rinsed 3X in TBS + then incubated in fluorescent secondary antibody diluted 1:1000 in TBS + at room temperature for 2–3 h with gentle agitation. Tissue was rinsed 3X with TBS, stained with DAPI, and mounted on slides in 0.15% porcine gelatin and allowed to dry in the dark overnight. Slides were then coverslipped with DPX mountant and imaged at 20X on a Zeiss LSM880 Airyscan Confocal Microscope or Nikon AX R confocal microscope. Quantification of cell marker overlap with GFAP-Cre-mCherry construct was conducted manually using the Colocalization plugin in Fiji (Version 2.9.0).