Animals were given a lethal dose of sodium pentobarbital (120 mg/kg) (MWI, 710101) and either enucleated immediately or perfused intracardially with 4% PFA. Eye were removed and fixed in PFA for 15–30 minutes. Following dissection, retinas were either kept whole or immersed in 30% sucrose overnight prior to freezing in TFM (EMS, 72592) and cryosectioning at 20 μm. Immunostaining of retinal whole-mounts and cryosections was conducted as described previously (Duan et al., 2014 (link); Krishnaswamy et al., 2015 (link)). Primary antibodies used: mouse anti-β-galactosidase (DSHB, 40-1a), rabbit anti-β-galactosidase (Duan et al., 2014 (link)), rabbit anti-calretinin (Millipore, AB5054), mouse anti-calretinin (Milipore, AB1568), goat anti-ChAT (Milipore, AB144P), goat anti-Chx10 (Santa Cruz Biotechnology, 21690), sheep anti-Chx10 (Exalpha, X118OP), mouse anti-cre (Milipore, AB3120), chicken anti-GFP (Abcam, ab13790), rabbit anti-Lhx3 (gift of Dr. Sam Pfaff) (Sharma et al., 1998 (link)), rabbit anti-mcherry (Krishnaswamy et al., 2015 (link)), rabbit anti-Nfia (Active Motif, 39397), Rabbit anti-Otx2 (Millipore, AB9566), mouse anti-PkarIIb (BD Bioscience, 610625), rabbit anti-PKC (Sigma, P4334), rabbit anti-Ppp1r17 (Atlas Antibodies, HPA047819), mouse anti-Syt2 (ZIRC, Znp-1). All secondary antibodies used were purchased from either Invitrogen or Jackson ImmunoResearch.
Ab13790
Manufactured by Abcam
Ab13790 is a laboratory reagent produced by Abcam. It is used for research purposes. Further details about its core function or intended use are not available.
Automatically generated - may contain errors
2 protocols using ab13790
1
Retinal Immunohistochemistry Techniques
2
Apoptosis Visualization in Anesthetized Larvae
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Terminally anesthetized larvae were fixed in 4% PFA in PBS for overnight at 4 °C. After removing the head and tail using micro scissors, larvae were permeabilized by subsequent incubation in acetone and proteinase K as described for whole-mount IF. Samples were re-fixed in 4% PFA in PBS and Click-iT TUNEL Alexa Fluor 647 Imaging Assay (Thermo Fisher Cat#C10247) was performed according to the manufacturer’s protocol to label apoptotic cells. Briefly, samples were equilibrated in TdT reaction buffer for 30 min at room temperature, followed by incubation in TdT reaction cocktail for overnight at room temperature. The click-it reaction was performed for 60 min at room temperature. Thereafter, samples were blocked in PBTx containing 3% bovine serum albumin and incubated over three nights with chicken anti-GFP antibody (Abcam Cat#ab13790; RRID:AB_300798, 1:500). After several washes in PBTx, samples were incubated over two to three nights with the secondary antibody of interest. Thereafter, samples were washed in PBTx and mounted in 75% glycerol in PBS. Imaging was done using a Plan-Apochromat 10x/0.45 M27 objective on a Zeiss LSM 980 confocal microscope.
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