Computational protocol: Spinster Homolog 2 (Spns2) Deficiency Causes Early Onset Progressive Hearing Loss

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Protocol publication

[…] The inner ears were rapidly dissected out and fixed in 4% paraformaldehyde at room temperature for 2 hours. The lateral walls were dissected out in PBS for surface preparation. Filamentous actin was visualized by rhodamine phalloidin (1∶200, Molecular Probe) at room temperature for 2 hours. Strial capillaries were visualized by Isolectin B4 (Vector Laboratories, 1∶50) at 4°C, overnight in PBS with 10% sheep serum). Samples were mounted with Vectashield Mounting medium (Vector, Cat. No: H-1000) and imaged by confocal microscopy (Carl Zeiss, LSM 510 META). The numbers of capillary branch points per field (220×220-µm fields) in the middle turn (40–70% of the distance along the cochlear duct from the base) of the stria vascularis (control, n = 4; homozygotes, n = 5, at P14. control, n = 3; homozygotes, n = 5, at P28) was quantified using image J. Data were presented as a density in a 100×100 µm field and statistics analysis was conducted using Mann-Whitney Rank Sum Test, SigmaPlot v12.0. Surface preparations were also used for analysis of Kcnq1 expression, using overnight incubation at 4°C with goat anti-Kcnq1 polyclonal antibody (Santa Cruz, 1∶200) followed by washing with PBS and incubation with donkey anti-goat secondary antibody (Invitrogen, 1∶500) prior to analysis by confocal microscopy. At least three homozygotes and three controls were used at P14, P28 and 6 months for phalloidin labeling to show marginal cell boundaries, and P14 and 5–6 weeks for Kcnq1 expression. The density of marginal cells was measured in the phalloidin-labelled whole mount preparations by counting the number of cells defined by their labeled boundaries in two areas each 100×100 µm from the middle turn (40–70%) of each cochlea (n = 4 homozygous mutants; 4 littermate controls). [...] Mice underwent ophthalmic screening at 15 weeks of age. They were assessed for gross morphological changes to the eye using a slit lamp (Zeiss SL130) and ophthalmoscope (Heine Omega 500). The eye was examined both undilated and dilated (topical tropicamide). Images using the slit lamp were collected using a Leica DFC420 camera. The mice were culled under terminal anaesthesia followed by cervical dislocation and both eyes from 3 male homozygous mutants and 3 wildtype mice were removed and fixed. Pupil-optic nerve sections were processed, stained with hematoxylin and eosin, and standard images were captured under light microscopy for review .For whole mount retinal analysis, heterozygotes and homozygotes were used (n = 3 for each genotype at P10, n = 2 at 8 weeks old) and the eyes removed and fixed in 4% PFA. Retinae were prepared and stained as described using flourescein-conjugated Griffonia simplicifolia Isolectin B4 (Vector Laboratories, UK) to label blood vessels, mouse anti-proteoglycan NG2 (Millipore UK Ltd., UK) to label pericytes, and donkey anti-rabbit secondary Alexa-594 (Molecular Probes, Life Technologies, UK). The homozygote and control mouse retinae were stained in the same well to control for changes in staining efficiency, distinguishing the retinae by different numbers of radial incisions. All tissues were mounted in Vectashield (Vector Laboratories Ltd., Peterborough, UK), imaged by confocal microscopy (Nikon A1R; Nikon Instruments, Inc., Melville, NY), and maximum intensity projections of z-stacks were created using NisElements AR Version 4.0 software (Nikon UK, Kingston Upon Thames, UK). The MetaMorph Angiogenesis Tube Formation application (Molecular Devices, Berkshire, UK) was used for quantification. Confocal images were used to determine the total area covered by vessels and by pericytes to calculate the percentage pericyte coverage of vessels and the total number of capillary branchpoints per unit area. We imaged three areas of each retina: three different regions of the central retina each encompassing an artery and vein, and two images each of peripheral arteries and peripheral veins, a total of five images/retina. Threshold values were kept the same for analysis of samples of the same stage. […]

Pipeline specifications

Software tools SigmaPlot, MetaMorph
Applications Miscellaneous, Laser scanning microscopy
Organisms Danio rerio, Mus musculus
Diseases Congenital Abnormalities, Brain Diseases, Retinal Degeneration