Journal: Cell Death Discovery

Article Title: Systemically administered peptain-1 inhibits retinal ganglion cell death in animal models: implications for neuroprotection in glaucoma

doi: 10.1038/s41420-019-0194-2

Figure Lengend Snippet: a αB-Crystallin (red) was detected in the nerve fiber layer (NFL) and in the outer plexiform layer (OPL) in the non-glaucomatous retinas. Only very weak immunostaining in the NFL was observed in the glaucomatous retinas. Representative images from the human retinas. The RGCs were labeled with a Brn3a antibody (green). The cell nuclei were stained with DAPI (blue). Scale bar = 50 μm. b The number of RGCs in the retinas of the CRYAB KO mice was lower than that in the retinas of the age-matched WT mice. The bar graphs represent the means ± SD of triplicate measurements. Scale bar = 200 μm. ns not significant, * p < 0.05 (unpaired t -test). GLC = ganglion cell layer

Article Snippet: After blocking buffer was removed and explants were incubated with goat anti-Brn3a (C-20) antibody (1∶500 dilution, Cat#. sc-31984, Santa Cruz Biotechnology, Dallas, TX) for 48 h at 4 °C.

Techniques: Immunostaining, Labeling, Staining

Journal: Cell Death Discovery

Article Title: Systemically administered peptain-1 inhibits retinal ganglion cell death in animal models: implications for neuroprotection in glaucoma

doi: 10.1038/s41420-019-0194-2

Figure Lengend Snippet: a Representative micrograms depicting the binding of the Cy5-peptain-1 to the surface and its internalization over time. b The graph shows the average change in intensity (average counts/ms) after 0, 30, and 60 min of incubation with Cy5-peptain-1 based on the changes shown in Fig. 2a. ns = not significant, **** p < 0.0001 (Tukey’s multiple comparison test). c and d peptain-1 protects primary rat RGCs from hypoxia-induced death. Primary RGCs were maintained either in a normoxic environment (10% CO 2 , 17.2% O 2 at 37 °C) or in a hypoxic environment (10% CO 2 and 0.5% O 2 at 37 °C). The cells were treated with either 12.5 μ g/ml peptain-1 or 12.5 μg/ml scrambled peptide (Scr-1). **** p < 0.0001 (Tukey’s multiple comparison test). n = 4 per group in two independent experiments. E, Peptain-1 protects adult rat RGCs in retinal explants from hypoxic conditions. The retinal explants were immunostained with a Brn3a antibody (green) to detect for RGCs. The bottom panels show the quantification of RGCs per mm 2 of the explant. Vehicle alone or Scr-1 induced no significant changes in the number of RGCs, but peptain-1 protected the RGCs. ns not significant, *** p < 0.001; **** p < 0.0001 (Tukey’s multiple comparison test). N = 4 explants per treatment

Article Snippet: After blocking buffer was removed and explants were incubated with goat anti-Brn3a (C-20) antibody (1∶500 dilution, Cat#. sc-31984, Santa Cruz Biotechnology, Dallas, TX) for 48 h at 4 °C.

Techniques: Binding Assay, Incubation, Comparison

Journal: Cell Death Discovery

Article Title: Systemically administered peptain-1 inhibits retinal ganglion cell death in animal models: implications for neuroprotection in glaucoma

doi: 10.1038/s41420-019-0194-2

Figure Lengend Snippet: a Fifty micrograms of a peptide (peptain-1 or Scr-2) was injected i.p. into either WT or CRYAB KO mice twice daily for 3 days following I/R injury. Fourteen days after injury, the number of Brn3a-positive RGCs (green) were counted in both the central and peripheral regions of the retina, and the numbers are shown in the bar graphs ( b ). ns not significant, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 (Tukey’s multiple comparison test). N = 4. Scale bar = 50 μm

Article Snippet: After blocking buffer was removed and explants were incubated with goat anti-Brn3a (C-20) antibody (1∶500 dilution, Cat#. sc-31984, Santa Cruz Biotechnology, Dallas, TX) for 48 h at 4 °C.

Techniques: Injection, Comparison