Journal: International Journal of Molecular Sciences
Article Title: Deletion of POMT2 in Zebrafish Causes Degeneration of Photoreceptors
doi: 10.3390/ijms232314809
Figure Lengend Snippet: EYS protein was reduced and mis-localized in pomt2 mutant zebrafish retinas. Retinal sections from zebrafish at 1-mpf were double immunostained with EYS (green fluorescence) and acetylated α-tubulin (red fluorescence). The sections were counterstained with DAPI to show nuclei. ( A , C ) Wild-type retina at 1-mpf. Most EYS-positive puncta in the wild-type retina were associated with the basal end of acetylated α-tubulin reactivity (arrows). ( B , D ) pomt2 sny5+13 homozygous mutant retina at 1-mpf. Overall, EYS immunoreactivity in the mutant was much lower than the wild-type. Most of the EYS-positive puncta were not associated with acetylated α-tubulin but were localized to the outer nuclear layer. ( E ) Counting of EYS-acetylated α-tubulin double staining from the outer nuclear layer to the retinal pigment epithelium (RPE) ( n = 3). Note that acetylated α-tubulin reactivity associated with EYS puncta were decreased in the mutant, and acetylated α-tubulin not associated with EYS puncta were increased in the mutant. ( F ) Percentage of EYS associated with α-tubulin immunoreactivity and not associated with acetylated α-tubulin ( n = 3). Note that EYS immunoreactivity associated with acetylated α-tubulin was reduced in pomt2 mutant retinas, but non-associated EYS puncta were increased in these animals. ( G ) Percentage of acetylated α-tubulin immunoreactivity associated and not associated with EYS ( n = 3). Note that acetylated α-tubulin immunoreactivity associated with EYS was reduced but acetylated α-tubulin immunoreactivity not associated with EYS was increased in pomt2 mutant retinas. Scale bar in ( D ): 10.5 µm for ( A , B ); 5 µm for ( C , D ).
Article Snippet: After fixation with 4% paraformaldehyde for 10 min, the sections were permeabilized with 0.1% Triton X-100 in phosphate buffer and incubated with 3% bovine serum albumin (BSA) in 0.1M phosphate buffer (PB) at room temperature for 1 h. Primary antibodies against GNAT2 (MBL International, Woburn, MA, USA, Cat#PM075, 1:400), EYS (Novus Biological, Centennial, CO, USA, Cat# NBP1-90038, 1:300), acetylated α-tubulin (Millipore-Sigma, Cat#T6793, 1:1000), red opsin (1D4, Abcam, Cat#AB5417, 1:1000), and rhodopsin (4D2, Abcam, Cat#AB98887, 1:300) were applied to the sections and incubated in a humidified chamber overnight at 4 °C.
Techniques: Mutagenesis, Fluorescence, Double Staining
Novus Biologicals is a verified supplier 
Novus Biologicals manufactures this product 
