Researcher builds new model to examine Ushersyndrome NatureComms
. After fixation the eyes were transferred to 30% sucrose, embedded in OCT medium, and stored at −80 °C until they were completely frozen then four micrometer-thick sections were cut. After 10 min fixation with acetone at −20 °C, the sections were dried for 10 min. Rehydration was done in 1x PBS, followed by 30 min in blocking solution . The sections were then incubated overnight at 4 °C with the primary antibodies diluted in a blocking solution.
. The insoluble interphotoreceptor cell matrix was then separated from the soluble IPM by centrifugation at 750×for 5 min. The supernatant was considered to contain soluble components of the IPM. The pellet was washed three times in 1x PBS, resuspended in 0.1x PBS with 2x protease inhibitors and homogenized. The suspension was centrifuged at 50,000×for 30 min in a Sorvall Discovery M150 ultracentrifuge equipped with a fixed angle rotor .
. Secondary antibodies against mouse and rabbit conjugated to HRP diluted 1:15,000 in 5% non-fat dry milk were used.Eyes were orientated via marking of the superior hemisphere along the vertical meridian at the limbus with a hot needle after euthanasia. A slit in the superior cornea was made and the eyes were then fixed for 2 h in 0.1 M sodium phosphate buffer containing 2.5% glutaraldehyde, 2% paraformaldehyde, and 0.025% CaClat 4 °C.
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