Journal: Neural Regeneration Research

Article Title: Necroptosis plays a crucial role in the exacerbation of retinal injury after blunt ocular trauma

doi: 10.4103/1673-5374.353848

Figure Lengend Snippet: Blunt ocular trauma induces retinal cell necroptosis. (A) Representative images of TUNEL staining (green) after blunt ocular trauma. The proportions of TUNEL-positive cells in the INL and ONL increased over time. Scale bar: 50 μm. (B) The numbers of TUNEL-positive cells in ONL and INL were recorded. Compared with the numbers of TUNEL-positive cells in the ONL and INL in the control group, the number of TUNEL-positive cells in the INL increased over time, while the number of TUNEL-positive cells in the ONL was sharply increased on the 14 th day after injury. (C) Necroptosis-related proteins were detected by western blotting assays, and the ratios of p-Rip3/Rip3 and p-MLKL/MLKL both increased over time. Data are shown as the mean ± SEM ( n = 5 per group). * P < 0.05, vs . control group (Student’s t -test). INL: Inner nuclear layer; MLKL: mixed lineage kinase domain-like protein; ONL: outer nuclear layer; Rip3: receptor-interacting protein kinase 3; TUNEL: TdT-mediated dUTP nick end labeling.

Article Snippet: Each membrane was blocked with PBS (pH 7.4) containing 10% bovine serum albumin for 1 hour, then incubated overnight at 4°C with one of the following primary antibodies (all diluted 1:1000 by antibody diluent (WB100D, NCM Biotech, Suzhou, China)): phospho-Rip3 (p-Rip3; rabbit; Cell Signaling Technology, Danvers, MA, USA; Cat# 91702), Rip3 (rabbit; Cell Signaling Technology; Cat# 95702), MLKL (rat; Millipore; Cat# MABC604, RRID: AB_2820284), phospho-MLKL (p-MLKL; mouse; Millipore; Cat# MABC1158), or β-actin (mouse; Proteintech; Cat# 66009-1-Ig, RRID: AB_2687938).

Techniques: TUNEL Assay, Staining, Western Blot, End Labeling