Retinal flat mounts immersing in marker solutions were processed to visualize the vascular basement membrane. Prior to immersion staining, one-fourth of the retinal tissue in each sample was incubated for 45 mins at room temperature in 5% normal bovine serum in PBS containing 0.5% Triton-X-100 (0.5% T-PBS). Subsequently, the flat mounts were incubated overnight at 4°C in a marker solution containing rabbit polyclonal anti-type IV collagen antibody solution (1 : 300; ab19808; Abcam, Cambridge, UK) for basement membrane [9 (link)]. Fluorescent goat anti-rabbit immunoglobulin (Ig) G (1 : 45; BA1105; Wuhan Boster Biological Technology, Ltd., Wuhan, China) was treated as a secondary antibody. Subsequent to secondary incubation at 20°C for 5 mins, the retinal flat mounts were washed three times in 0.5% T-PBS, kept into DAPI for 5 mins, and washed another three times in 0.5% T-PBS. Then, the retinal flat mounts were prepared in a Vectashield (Wuhan Boster Biological Technology, Ltd.) and analyzed using a Zeiss LSM 710 confocal laser scanning microscope to determine the area and number of retinal neurocytes and the number of type-IV collagen strands.
Vectashield
Manufactured by Wuhan Boster Biological Technology
Vectashield is a mounting medium used to preserve fluorescent signals in microscopy applications. It is designed to minimize photobleaching and maintain the integrity of fluorescent labels during imaging.
Automatically generated - may contain errors
2 protocols using vectashield
1
Visualizing Retinal Vascular Basement Membrane
2
Retinal Vascular Basement Membrane Visualization
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Retinal flat-mounts were processed to visualize the vascular basement membrane by immersing them in marker solutions. Prior to immersion staining, the retinal flat-mounts were incubated for 30 min at room temperature in 5% normal bovine serum in PBS containing 0.5% Triton-X-100 (0.5% T-PBS) as a blocking agent. Subsequently, the flat-mounts were immersed overnight at room temperature in a marker solution containing rabbit polyclonal anti-type IV collagen antibody solution (1:300; ab19808; Abcam, Cambridge, UK) for basement membrane (19 (link)). Fluorescent goat anti-rabbit immunoglobulin (Ig) G (1:45; BA1105; Wuhan Boster Biological Technology, Ltd., Wuhan, China) was used as a secondary antibody. Subsequent to secondary incubation at 20°C for 5 min, the retinal flat-mounts were washed three times in 0.5% T-PBS, placed into DAPI for 5 min and washed an additional three times in 0.5% T-PBS. The retinal flat-mounts were then mounted on a Vectashield (Wuhan Boster Biological Technology, Ltd.) and analyzed using a Zeiss LSM 710 confocal laser scanning microscope to determine the number of type-IV collagen strands.
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