Ab13970

Ultrathin sections of LR White-embedded tissue were immunostained for srAT. Ultramicrotomy exposed epitopes at the section surface. Thus, sections could be stained, even though antibodies do generally not penetrate the resin.
A detailed protocol can be found in (Markert et al., 2017 ). In brief, sections were placed in a humid chamber, and blocking buffer was applied to the sections prior to staining with primary and secondary antibodies. They were washed with buffer and then stained with a DNA stain, where applicable. Lastly, sections were mounted with Mowiol and stored at 4°C for up to a week before fluorescence imaging.
We used a polyclonal antibody against GFP (Abcam, catalog number: ab13970) at a dilution of 1:500 and a polyclonal antibody against FUS (Bethyl, A300-293A) at a dilution of 1:1000.

Immunofluorescence staining was done 24 or 48 h after transfection or electroporation. Cells were rinsed once with 1x PBS and fixed with 4% PFA at room temperature for 15 min. Fixed cells or brain slices were rinsed with 1x PBS, incubated with blocking buffer (1x PBS with 0.3% Triton-X-100 and 5% donkey serum) at room temperature for 30 min, and further incubated with primary antibodies diluted in blocking buffer overnight at 4 °C. After three brief rinses in 1x PBS, slides were incubated for 1 h at room temperature with fluorophore-conjugated secondary antibodies (Donkey anti-chicken 488 (Jackson ImmunoReseach, 703-546-155), donkey anti-mouse 488 (Thermo Scientific, A21202), donkey anti-rabbit 594 (Thermo Scientific, A21207), donkey anti-rat 647 (Thermo Scientific, A48272); 1:1000 dilution for all). Slides were scanned with a Leica SP5 inverted confocal microscope. The following primary antibodies were used: anti-HA (Millipore 3F10 clone, rat, 1:1000), anti-GFP (Abcam ab13970, chick, 1:2000), anti-PUM2 (Bethyl A300-202A, rabbit, 1:1000), anti-CD24-PE (BioLegend 138504, rat, 1:1000), anti-CD133-APC (BioLegend 141208, rat, 1:1000), anti-PAX6 (Covance PRB-278P, rabbit, 1:500), anti-APP (BioLegend 802803, mouse, 1:1000).