Anti aldh1l1

Formalin-fixed brain halves divided at the midline (left hemisphere) were treated in formic acid (95%) to deactivate prion infectivity before being embedded in paraffin. 4 μm sections mounted on slides were processed for hematoxylin-eosin (H&E) staining and immunohistochemistry. To expose epitopes, slides were subjected to 20 min hydrated autoclaving at 121°C in trisodium citrate buffer, pH 6.0 with 0.05% Tween 20. For detection of disease-associated PrP, 5 min treatment with 88% formic acid was used following autoclaving. PrP was stained with anti-prion antibody SAF-84 (Cayman Chemical, Ann Arbor, MI). Rabbit anti-Iba1 (Wako, Richmond, VA) was used to stain microglia. Rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) (Promega, Madison, WI), chicken polyclonal anti-GFAP (Sigma-Aldrich, St. Louis, MO), rabbit polyclonal anti-Aldh1l1 (Abcam, Cambridge, MA), and rabbit monoclonal anti-S100ß (Abcam, Cambridge, MA) were used to stain astrocytes. Detection was performed using DAB Quanto chromogen and substrate (VWR, Radnor, PA).

The protocols used for immunofluorescence are described elsewhere [7 (link)]. Anti-GASC1, anti-ALDH1L1, anti-NOTCH1 (Abcam, USA), anti-H3K9Me2, and anti-H3K9me3 (Millipore, USA) antibodies were used as primary antibodies. Donkey anti-rabbit IgG (Alexa Fluor® 488), Donkey anti-rabbit IgG (Alexa Fluor® 555), donkey anti-sheep IgG (Alexa Fluor® 488), and goat anti-mouse IgG (Alexa Fluor® 488) (Abcam, USA) were used as secondary antibodies. Nuclear staining was performed with 40,6-diamidino-2-phenylindole (DAPI; 1:1000; Roche, USA).

Cortical and cerebellar astrocytes were seeded on poly-L-lysine pre-coated coverslips in 24-well plates at a density of 3 × 10⁴ cells/well in the culture medium. When they reached 70% confluence, the cells were fixed with 10% neutral buffered formalin for 15 min at room temperature. Cortical and cerebellar astrocytes were permeabilized with PBST (containing 0.2% Trion X, pH 7.4). Cells were blocked with 3% donkey serum in superblock buffer for 1 h at room temperature and incubated with the following primary antibodies overnight at 4 °C (anti-GFAP, 1:200, Cell Signaling Technology, Cat #3670S; anti-Iba1, 1:100, Cell Signaling Technology, Cat #17198; anti-ALDH1L1, 1:100, Abcam, Cat #87117). The cells were then incubated with secondary antibodies (Alex-Fluor^TM 488 donkey anti-mouse IgG, 1: 500, Cat A21202; Alex-Fluor^TM 568 donkey anti-rabbit IgG, 1: 500, Cat: # A10042) for 60 min at room temperature and mounted with ProLong^TM Gold antifade reagent with DAPI (Lot #1899752, Invitrogen, Waltham, MA, USA). Fluorescence and differential interference contrast (DIC) images were taken with a Zeiss Axio Observer Z1 microscope (Carl Zeiss, Dublin, CA, USA). The morphologies of cortical and cerebellar astrocytes were analyzed using FIJI-ImageJ (RRID:SCR_002285, NIH, Bethesda, MD, USA), and a Simple Neurite Tracer (SNT) plugin [24 (link)].