Ga50461 2

After 1–2 days in culture, except where noted, wt and GCaMP6f-expressing murine ACC were rinsed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde in PBS for 10 min. Cultures were blocked in PBS containing 0.1% Triton X-100 (PBST) and 10% fetal bovine serum (FBS) and incubated overnight at 4°C with antibodies against TH (rabbit, PA5-85167, ThermoFisher, Waltham, MA, USA), phenylethanolamine N-methyltransferase (PNMT) (rabbit, AB110, MilliporeSigma, Burlington, MA, USA), or S100β (rabbit, GA50461-2, Agilent, Santa Clara, CA, USA). For GCaMP6f-expressing cells, the overnight incubation included a green fluorescent protein (GFP) antibody (goat, 600-101-215, Rockland Immunochemicals, Pottstown, PA, USA). Primary antibodies were diluted 1:1000. Cells were then rinsed several times with PBS and incubated for one hour at room temperature in the dark with AlexaFluor 594-conjugated donkey-anti-rabbit and/or AlexaFluor 488-conjugated donkey anti-goat antibodies (ThermoFisher). Secondary antibodies were diluted 1:500 in 10% FBS in PBST that also contained 1 μg/ml bisbenzimide (Hoechst 33342; ThermoFisher) to label nuclei. Cells were rinsed with PBS and brightfield and fluorescence images were acquired using a Nikon TE 2000 epifluorescence microscope equipped with an iXonEM + DU-897 EMCCD camera (Andor).

Immunofluorescence staining was performed by using paraffinized slices as described previously.⁴⁷ Sagittal sections of mouse brain were stained with anti‐GFAP (Z033401‐2, Agilent Technologies, CA, USA) and anti‐S100β (GA50461‐2, Agilent Technologies) antibodies for astrocytes and an anti‐IBA1 antibody (Fujifilm, Tokyo, Japan) for microglia. Stained slices were mounted with DAPI‐Fluoromount‐G (SouthernBiotech). Fluorescence was acquired by using the Axiocam 506 mono (Carl Zeiss, Oberkochen, Germany) equipped with an inverted 63× 0.45NA UPLFL objective on an Axio Imager M2 Upright Microscope (Carl Zeiss). Images were taken in the channel sequence of FITC (Ex 493, Em 528), DAPI (Ex 390, Em 435), and Texas Red (Ex 576, Em 603). Six to ten locations in each slide were selected for the quantification. To examine astrocyte morphology/hypertrophy, GFAP signal area for individual astrocytes was measured as cell size, and the average area for each cell process was measured to determine GFAP fiber thickness. To measure astrocyte cell number, S100β⁺ cells were counted in cortex and hippocampus images.