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 mikrodissektion för enkelkärnesekvensering eller immunfärgning

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.

Författare i fokus: Dissektion av Stria vascularis hos vuxna möss för enkelkärnesekvensering eller immunfärgning  

Dissektion av vuxen mus Stria vascularis för sekvensering av en kärna eller immunfärgning

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  

Immunfärgning av mus Stria vascularis och vävnadsmontering

Förbered dig för immunfärgning genom att dissekera stria vascularis från musen och tillsätt vävnaden till en 24-brunnsplatta innehållande 200 mikroliter 4% paraformaldehyd i 1X PBS. Inkubera vävnaden i 20 minuter vid rumstemperatur. utföra två korta tvättar på 1X PBS på en orbitalskakare vid lågt varvtal.Ta bort PBS, utför sedan permeabilisering och blockering i minst en timme vid rumstemperatur i 300 mikroliter PBST-lösning. Färga sedan vävnaden med den primära antikroppen över natten vid fyra grader Celsius. Tillsätt sedan en sekundär antikropp och inkubera i två timmar vid rumstemperatur på en orbitalskakare vid lågt varvtal.Täck plattan för att skydda den fluorescerande märkta sekundära antikroppen från ljus. Förbered sedan en större mikroglid genom att placera två små limstreck längs 25 mm-axeln, bara mindre än 18 x 18 millimeter glasskydd. Placera en droppe monteringsreagens mellan limstrimmorna.Använd nummer 55 pincett, gab så lite vävnad som möjligt i ena änden av SV och överför den från PBS till monteringsreagenset. Placera ena änden av lockslipen på bilden där en limstrimma placerades. Släpp sedan försiktigt locket för att undvika att skapa luftbubblor.Försegla fästet genom att badda en droppe transparent nagellack i varje hörn av fästet. Märk provet på glasluckan, bort från visualiseringsfältet. Visualisera SV-vävnaden under ett dissektionsmikroskop för att säkerställa att den ligger platt och inte nära några luftbubblor.Om SV-vävnaden inte är korrekt orienterad, försök att trycka ut luftbubblor eller försiktigt ta bort det mindre glaslocket och flytta SV. Ett helt fäste av SV från P30-mus förbereddes och konfokal avbildning utfördes. ZsGreen-uttryck observerades i mellancellerna, GSIB4 märkta endotelceller och DAPI märkte kärnorna.