Name: Immunostaining of Mouse Stria Vascularis and Tissue Mounting
Uploaded: 2023-04-21T12:50:00
Description: Watch this Scientific Journal Video about Immunostaining of Mouse Stria Vascularis and Tissue Mounting at JoVE.com

immunostaining of mouse stria vascularis and tissue mounting

Microdisección de estrías vasculares para secuenciación de un solo núcleo o inmunotinción

The cochlea consists of three fluid filled chambers, the scala vestibuli, scala media, and scala tympani. The scala vestibuli and scala tympani each contain perilymph, which has a high concentration of sodium (138 mM) and a low concentration of potassium (6.8 mM)1. The scala media contains endolymph, which has a high concentration of potassium (154 mM) and a low concentration of sodium (0.91 mM)1,2,3. This difference in ion concentration can be referred to as the endocochlear potential (EP), and is primarily generated by the movement of potassium ions through various ion channels and gap junctions in the stria vascularis (SV) along the lateral wall of the cochlea4,5,6,7,8,9,10,11. The SV is a heterogenous, highly vascularized tissue that lines the medial aspect of the lateral wall of the cochlea and contains three main cell types: marginal, intermediate, and basal cells12 (Figure 1).
Marginal cells are connected by tight junctions to form the most medial surface of the SV. The apical membrane faces the endolymph of the scala media and contributes to potassium ion transport into the endolymph using various channels, including KCNE1/KCNQ1, SLC12A2, and Na+-K+-ATPase (NKA)5,10,13,14. Intermediate cells are pigmented cells that reside between marginal and basal cells and facilitate potassium transport through the SV using KCNJ10 (Kir 4.1)15,16. Basal cells lie in close proximity to the lateral wall of the cochlea and are closely associated with fibrocytes of the spiral ligament to promote potassium recycling from the perilymph12. Pathology of the SV has been implicated in numerous otologic disorders17,18. Mutations in genes expressed in the major SV cell types, such as Kcnq1, Kcne1, Kcnj10, and Cldn11, can cause deafness and SV dysfunction, including the loss of EP19,20,21,22,23. In addition to the three major cell types, there are other less-studied cell types in the SV, such as spindle cells22, root cells12,24, macrophages25, pericytes26, and endothelial cells27, that have incompletely defined roles involving ionic homeostasis and the generation of EP28.
In comparison to bulk RNA-sequencing, single-nuclei RNA-sequencing (sNuc-Seq) provides information about cell heterogeneity, rather than an average of mRNA across a group of cells29, and can be particularly useful when studying the heterogenous SV30. For example, sNuc-Seq has produced transcriptional analysis that suggests there may be a role for spindle and root cells in EP generation, hearing loss, and Meniere&#39;s disease18. Further transcriptional characterization of the various SV cell types can provide us with invaluable information on the pathophysiology underlying different mechanisms and subtypes of SV-related hearing fluctuation and hearing loss. The harvest of these delicate inner ear structures is of paramount importance to optimal tissue analysis.
In this study, the microdissection approach to access and isolate the stria vascularis from the adult mouse cochlea for sNuc-Seq or immunostaining is described. Dissection of the adult mouse SV is required to understand various SV cell types and further characterize their role in hearing.

Autor destacado: Disección de estrias vasculares de ratones adultos para secuenciación de un solo núcleo o inmunotinción  

Disección de estrías vasculares de ratón adulto para secuenciación de un solo núcleo o inmunotinción

The stria vascularis functions to generate the endocochlear potential and regulate ion homeostasis in the cochlea. The auditory development and restoration program seeks to understand the underlying mechanisms connecting dysfunctional ion homeostasis with hearing and stability disorders, which are diseases where either fluctuation in or sudden hearing loss occurs. Work in my lab has established the transcriptional landscape of the stria vascularis and has contributed to our understanding of its importance in hearing.We have used these data to draw parallels to poorly-understood hearing and stability disorders, including Meniere's disease. This protocol enables the dissection and isolation of whole-mount stria vascularis for further molecular studies including immunohistochemistry. Given that stria vascularis cell types are intricately intertwined with one another, the use of nuclear isolation and single-nucleus RNA sequencing has facilitated our understanding of the distinct transcriptional machinery involved.

Watch this Scientific Journal Video about Immunostaining of Mouse Stria Vascularis and Tissue Mounting at JoVE.com

Immunostaining of Mouse Stria Vascularis and Tissue Mounting  

Inmunotinción de estrías vasculares de ratón y montaje tisular

Prepárese para la inmunotinción diseccionando la estría vascular del ratón y agregue el tejido a una placa de 24 pocillos que contiene 200 microlitros de paraformaldehído al 4% en 1X PBS. Incubar el tejido durante 20 minutos a temperatura ambiente. realizar dos lavados cortos de PBS 1X en un agitador orbital a bajas RPM.Retire el PBS, luego realice la permeabilización y el bloqueo durante un mínimo de una hora a temperatura ambiente en 300 microlitros de solución de PBST. Luego, tiñe el tejido con el anticuerpo primario durante la noche a cuatro grados centígrados. Luego agregue un anticuerpo secundario e incube durante dos horas a temperatura ambiente en un agitador orbital a bajas RPM.Cubra la placa para proteger de la luz el anticuerpo secundario marcado con fluorescencia. A continuación, prepare un microportaobjetos más grande colocando dos pequeñas rayas de pegamento a lo largo del eje de 25 mm, un poco más pequeño que el deslizamiento de cubierta de vidrio de 18 por 18 milímetros. Coloque una gota de reactivo de montaje entre las rayas de pegamento.Usando pinzas número 55, gabe la menor cantidad de tejido posible en un extremo del SV y transfiéralo de PBS al reactivo de montaje. Coloque un extremo del cubreobjetos en la corredera donde se colocó una raya de pegamento. Luego suelte suavemente el deslizamiento de la cubierta para evitar crear burbujas de aire.Selle el soporte frotando una gota de esmalte de uñas transparente en cada esquina del soporte. Etiquete la muestra en el resbalón de la cubierta de vidrio, lejos del campo de visualización. Visualice el tejido SV bajo un microscopio de disección para asegurarse de que esté acostado y no cerca de ninguna burbuja de aire.Si el tejido SV no está orientado correctamente, intente expulsar las burbujas de aire o retire con cuidado el deslizamiento de la cubierta de vidrio más pequeño y vuelva a colocar el SV. Se preparó una montura completa de SV del ratón P30 y se realizaron imágenes confocales. La expresión de ZsGreen se observó en las células intermedias, GSIB4 marcó las células endoteliales y DAPI marcó los núcleos.