The eyes were enucleated and fixed with 4% paraformaldehyde, and the whole retina was isolated and incubated in 3% crude trypsin (Gibco, Grand Island, NY) containing 200mM sodium fluoride at 37°C for 45–70 minutes. The neuroretinal tissue was gently brushed away under a microscope, and the vasculature was stained with terminal deoxyribonucleotide TUNEL using an In Situ Cell Death Kit (Cat. No. 11684795910, Roche Molecular Biochemicals). As a positive control, retinal vasculature treated with DNAse was also stained with TUNEL. The TUNEL-positive capillary cells were counted under a fluorescence microscope, and the microvasculature was then stained with periodic acid Schiff-hematoxylin to count acellular capillaries by light microscopy [22 (link), 26 (link)].
Protocol detail
Retinal Vascular Trypsin Assay
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Capillaries
Cell death
Cells
Deoxyribonucleotide
Dnase
Eyes
Fluorescence microscope
Light microscopy
Microscope
Microvasculature
Paraformaldehyde
Periodic acid
Retina
Retinal vasculature
Sodium fluoride
Tissue
Trypsin
Tunel
Corresponding Organization :
Other organizations : Wayne State University
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Variable analysis
independent variables
- Incubation time (45-70 minutes)
- Number of TUNEL-positive capillary cells
- Number of acellular capillaries
- Fixation with 4% paraformaldehyde
- Trypsin concentration (3% crude trypsin)
- Trypsin buffer (200mM sodium fluoride)
- Incubation temperature (37°C)
- Positive control: Retinal vasculature treated with DNAse and stained with TUNEL
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