cryosectioning-and-immunostaining-of-mouse-retina

Cryosectioning and Immunostaining of Mouse Retina

Tissue sectioning and immunohistochemistry are essential techniques in histological and pathological studies of retinal diseases using animal models. ...

University of Electronic Science and Technology of China

Retinitis pigmentosa (RP) is a complex group of hereditary retinal dystrophies. Although >60 genes have been identified to be associated with non‑syndromic RP, the exact genetic variant remains elusive in numerous cases of RP. In the present study, a Chinese pedigree affected by RP with autosomal recessive inheritance, including a total of seven members with one affected patient and six unaffected individuals, was recruited. Comprehensive ophthalmic examinations were performed on the proband and the proband's unaffected daughter. Genomic DNA was extracted from peripheral blood. Whole‑exome sequencing (WES) was performed for the affected individual. The candidate pathogenic variant was verified by direct Sanger sequencing. The affected individual presented with classical clinical symptoms of RP. A novel homozygous variant, c.265delC (p.L89Ffs*3) in the cyclic nucleotide‑gated channel subunit α 1 gene was identified in the affected patient. This homozygous variant was absent in other unaffected family members and 600 ethnicity‑matched healthy controls. The variant was co‑segregated with the disease phenotype in an autosomal recessive manner.

Identification of a novel homozygous variant in the CNGA1 gene in a Chinese family with autosomal recessive retinitis pigmentosa.

Immunohistochemistry using mouse retinal cryosections is a routine assay used in vision research. However, retinal tissues are fragile, and it is difficult to obtain an ideal retinal cryosection. Here, we developed a modified method for preparing retinal cryosection. Super Glue was applied on the surface of the sclera before the cornea and the lens are removed from either the unfixed or PFA-fixed mouse eyeballs. The new methods largely prevented retinal detachment in mouse retinal cryosections. Immunostaining of retinal cryosections derived from PFA-fixed mouse eyes using rod and cone markers yielded high-quality immunofluorescent images. Immunolabeling of retinal cryosections obtained from unfixed mouse eyes using a cilium-specific marker had improved orientations of photoreceptor connecting cilia. This new method substantially improves the morphology and immunostaining results of fixed and unfixed mouse eyes.

Article	Year
Identification of a novel homozygous variant in the CNGA1 gene in a Chinese family with autosomal recessive retinitis pigmentosa. Molecular medicine reports\| PubMed ID: 32705276	2020
An improved method for preparation of mouse retinal cryosections. European journal of histochemistry : EJH\| PubMed ID: 32880132	2020

Yongqiong Lin

Biography

JoVE Journal Publications

Other Publications