Ab6662

The histology and immunostaining were conducted as previously described (Xie et al., 2018 ; Tang et al., 2016a (link)). After pentobarbital overdose, mice were intracardially perfused with 0.9% saline solution followed by 4% paraformaldehyde in PBS. Mouse brains were removed immediately and placed in 4% paraformaldehyde solution overnight for post-fixation and then soaked in 30% sucrose solution for at least 48 h for cryoprotection. With a cryostat (Leica CM1950), the brains were sequentially cut into 40-μm-thick coronal sections from the olfactory bulb to the cerebellum (approximately +4.50 mm to −6.40 mm from the bregma). The sections were cleaned with PBS and mounted on chrome-gelatin subbed glass slides in sequence. To enhance the fluorescence intensity of the HSV-EGFP labeled slice, the sections were blocked with 3% BSA in PBS-0.3% Triton X-100 for 1 h at 37°C and subsequently incubated with anti-GFP antibody conjugation FITC (1:400, Abcam, ab6662) for 2 h at 37°C. After washing, brain sections were coverslipped with 70% DAPI-glycerol mounting medium.

Mice were euthanized by cervical dislocation. Left hemispheres were removed and fixed for 72 h in 4% paraformaldehyde (PFA) in PBS. Forty-micrometer coronal sections were obtained using a Leica vibratome (Leica VT1000S). Next, free floating sections were washed three times in PBS, treated with NHCl for 30 min, and washed again three times with PBS. Floating sections were permeabilized and blocked with PBS containing 3% Triton X-100, 0.02% Azide, 2% BSA, and 3% NGS (Ab buffer) for 1 h at RT. After three washes in PBS, brain slices were incubated overnight at 4 °C with FITC-conjugated GFP primary antibody (AB6662; 1:500, Abcam) for signal enhancement. Sections were washed three times and incubated for 2 h at RT with fluorescent secondary antibody Alexa Fluor 488 goat anti-mouse (1:400; from Jackson Immuno Research, West Grove, PA, USA). Sections were analyzed using a two-photon confocal microscope (Leica SP5).

To assess AR upregulation in Tg(gstp1:GFP) fish, larvae 4 hr post light illumination or bolus LDE treatment were dechorionated, washed twice in ice-cold PBS and fixed in 4% paraformaldehyde in 1 X PBS (Gibco 14190169) for at least overnight with gentle rocking at 4 °C. Fixed larvae were permeabilized with chilled methanol at –20 °C for 4 hr–overnight. Fish were then washed 2 times with PBS-0.1%Tween-1% DMSO for 30 min each with gentle rocking, then blocked in PBS-0.1%Tween containing 2% BSA and 10% FBS, then stained with anti-GFP FITC conjugated (Abcam, ab6662) primary antibody overnight at 4 °C in blocking buffer. Subsequently, the larvae were washed twice (30 min each wash), re-blocked for 1 hr at room temperature, and incubated with the AlexaFluor 568-conjugated fluorescent secondary antibodies (Abcam, ab175707) in blocking buffer for 1.5 hr at room temperature with gentle rocking, and then washed three times. Fish were imaged on 2% agarose plates on a Leica M205-FA equipped with a stereomicroscope. Quantitation of IF data was performed using ImageJ/FIJI (NIH).
To assess protein expression in zebrafish, larvae were fixed at 34 hours post fertilization (hpf) after dechorionation. Permeabilization and immunostaining protocols are as above except antibodies to the desired protein/tag and appropriate secondary antibodies were used.