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

Stria vascularis mikrodisseksjon for enkeltkjernesekvensering eller immunfarging

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.

Forfatter Spotlight: Disseksjon av voksen mus Stria Vascularis for enkeltkjernesekvensering eller immunfarging  

Disseksjon av voksen mus stria vascularis for enkeltkjernesekvensering eller immunfarging

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  

Immunfarging av mus stria vascularis og vev montering

Forbered deg på immunfarging ved å dissekere stria vascularis fra musen og tilsett vevet til en 24-brønnsplate som inneholder 200 mikroliter 4% paraformaldehyd i 1X PBS. Inkuber vevet i 20 minutter ved romtemperatur. utføre to korte vasker av 1X PBS på en orbital shaker ved lavt turtall.Fjern PBS, og utfør deretter permeabilisering og blokkering i minst en time ved romtemperatur i 300 mikroliter PBST-oppløsning. Deretter flekker vevet med det primære antistoffet over natten ved fire grader Celsius. Deretter tilsettes et sekundært antistoff og inkuberes i to timer ved romtemperatur på en orbital shaker ved lavt turtall.Dekk platen for å beskytte det fluorescerende merkede sekundære antistoffet mot lys. Forbered deretter et større mikrolysbilde ved å plassere to små striper lim langs 25 mm-aksen, bare mindre enn glassdekselet på 18 x 18 millimeter. Plasser en dråpe monteringsreagens mellom limstripene.Bruk nummer 55 tang, gab så lite vev som mulig på den ene enden av SV, og overfør det fra PBS til monteringsreagenset. Plasser den ene enden av dekselet på lysbildet der en stripe lim ble plassert. Slipp deretter dekselet forsiktig for å unngå å skape luftbobler.Forsegle festet ved å dryppe en dråpe gjennomsiktig neglelakk på hvert hjørne av festet. Merk prøven på glassdekselet, vekk fra visualiseringsfeltet. Visualiser SV-vevet under et disseksjonsmikroskop for å sikre at det ligger flatt og ikke i nærheten av luftbobler.Hvis SV-vevet ikke er riktig orientert, kan du prøve å skyve ut luftbobler eller forsiktig fjerne det mindre glassdekselet og flytte SV. En hel montering av SV fra P30 mus ble klargjort og konfokal avbildning ble utført. ZsGreen-ekspresjon ble observert i mellomliggende celler, GSIB4-merkede endotelceller og DAPI merket kjernene.