vectashield dapi aqueous mounting medium  (Vector Laboratories)

Journal: Biomolecules

Article Title: Inherited Retinal Degeneration: Towards the Development of a Combination Therapy Targeting Histone Deacetylase, Poly (ADP-Ribose) Polymerase, and Calpain.

doi: 10.3390/biom13040581

Figure Lengend Snippet: Figure 1. Effects of mono- and combination therapies on photoreceptor cell death. Retinal explant cultures derived from either wild-type (wt) or rd1 animals were treated until post-natal day 11 with specific inhibitors targeting calpain (CAST), HDAC (SAHA), or PARP (OLA), or were treated with combinations of these inhibitors. (A) Retinal cross-sections obtained from treated or untreated (Untr.) specimens were stained with the TUNEL assay (magenta) to detect dying cells. DAPI (grey) was used as nuclear counterstain. (B) Normalized quantification (rd1 max value = 1, wt min value = 0) of cell death in the outer nuclear layer (ONL) in the various treatment groups. Note the strong increase in ONL cell death in rd1 retina compared to wt and the decreased cell death rates afforded by the various treatments. Combined treatment with either SAHA + CAST or OLA + CAST did not produce additional rescue effects compared to single drug treatment. Images shown represent at least six different specimens for each genotype. Untr. wt: 12 explants from 12 different mice; untr. rd1: 27/27; CAST rd1: 14/14; SAHA rd1: 18/18; OLA rd1: 17/17; SAHA + CAST rd1: 6/6; OLA + CAST rd1: 15/15; error bars represent SD; INL = inner nuclear layer; GCL = ganglion cell layer. Scale bar = 50 µm; significance levels: **, p < 0.01; ****, p < 0.0001.

Article Snippet: Finally, the sections were washed twice with PBS, mounted using Vectashield with DAPI (ab104139; Abcam, Cambridge, UK), and imaged under a Zeiss ApoTome 2 microscope for further analysis.

Techniques: Derivative Assay, Staining, TUNEL Assay

Journal: Biomolecules

Article Title: Inherited Retinal Degeneration: Towards the Development of a Combination Therapy Targeting Histone Deacetylase, Poly (ADP-Ribose) Polymerase, and Calpain.

doi: 10.3390/biom13040581

Figure Lengend Snippet: Figure 4. Effects of mono- and combination therapies on photoreceptor PARP activity. (A) Retinal PARP activity was assessed using an in situ activity assay (green). DAPI was used as nuclear coun- terstain (grey). In the outer nuclear layer (ONL) of the untreated (Untr.) rd1 retina, PARP activity was strongly increased compared to the wild-type (wt). (B) Quantification of the numbers of PARP activity-positive cells in the various experimental groups. While the calpain inhibitor CAST had no effect on PARP activity, it was significantly reduced by both the HDAC inhibitor SAHA and the PARP inhibitor. Combined treatment with SAHA and CAST or OLA and CAST reduced PARP activity to levels comparable to monotherapy. Images shown represent at least six different specimens for each genotype. Untr. wt: 9 explants from 9 different mice; untr. rd1: 20/20; CAST rd1: 10/10; SAHA rd1: 15/15; OLA rd1: 9/9; SAHA + CAST rd1: 6/6; OLA + CAST rd1: 8/8; error bars represent SD; INL = inner nuclear layer; GCL = ganglion cell layer. Scale bar = 50 µm; significance levels: ***, p < 0.001; ****, p < 0.0001.

Article Snippet: Finally, the sections were washed twice with PBS, mounted using Vectashield with DAPI (ab104139; Abcam, Cambridge, UK), and imaged under a Zeiss ApoTome 2 microscope for further analysis.

Techniques: Activity Assay, In Situ