Immunohistochemical Analysis of S-Opsin Gradient
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Corresponding Organization : University of Akron
Other organizations : Brown University
Protocol cited in 6 other protocols
Variable analysis
- Criteria for dissection quality - Any retina that was appreciably torn in such a way as to mimic the appearance of a reliving cut, or any retina that exhibited significant tissue loss or damage of the central or mid-central retina was discarded.
- Observation of the s-opsin gradient in dissected retinas
- Mounting of dissected retinas on nitrocellulose membrane paper
- Immunohistochemistry protocol similar to Sondereker et al. (2017)
- Retinas fixed in 4% paraformaldehyde for 40 minutes
- Retinas rinsed in 0.1M phosphate-buffered saline (PBS)
- Retinas placed in blocking solution and incubated at 4°C overnight
- Retinas incubated at 4°C in blocking solution for six days with primary antibody goat anti-s-opsin
- Retinas washed in 0.1M PBS and placed in blocking solution overnight at 4°C with secondary antibody donkey anti-goat Alexa Fluor 594
- Retinas rinsed in 0.1M PBS before being mounted on a glass slide with Aquamount and covered with a 1.5 μm thick coverslip
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