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

Microdissection de stries vasculaires pour le séquençage d’un seul noyau ou l’immunocoloration

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.

Pleins feux sur l’auteur : Dissection de stria vascularis de souris adultes pour le séquençage d’un seul noyau ou l’immunomarquage  

Dissection de la strie vasculaire de souris adulte pour le séquençage mononucléaire ou l’immunomarquage

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  

Immunomarquage de la strie vasculaire de souris et montage tissulaire

Préparez-vous à l’immunomarquage en disséquant la strie vasculaire de la souris et ajoutez le tissu dans une plaque de 24 puits contenant 200 microlitres de paraformaldéhyde à 4% dans 1X PBS. Incuber le tissu pendant 20 minutes à température ambiante. effectuer deux courts lavages de 1X PBS sur un agitateur orbital à bas régime.Retirez le PBS, puis effectuez la perméabilisation et le blocage pendant au moins une heure à température ambiante dans 300 microlitres de solution PBST. Ensuite, colorez le tissu avec l’anticorps primaire pendant la nuit à quatre degrés Celsius. Ajoutez ensuite un anticorps secondaire et incuber pendant deux heures à température ambiante sur un agitateur orbital à bas régime.Couvrir la plaque pour protéger de la lumière l’anticorps secondaire marqué par fluorescence. Ensuite, préparez une microlame plus grande en plaçant deux petites traînées de colle le long de l’axe de 25 mm, juste plus petites que le couvercle en verre de 18 par 18 millimètres. Placez une goutte de réactif de montage entre les traînées de colle.À l’aide d’une pince numéro 55, utilisez le moins de tissu possible à une extrémité du SV et transférez-le du PBS au réactif de montage. Placez une extrémité du couvercle sur la glissière où une traînée de colle a été placée. Relâchez ensuite doucement le couvercle pour éviter de créer des bulles d’air.Scellez le support en tamponnant une goutte de vernis à ongles transparent sur chaque coin du support. Étiquetez l’échantillon sur le couvercle en verre, loin du champ de visualisation. Visualisez le tissu SV sous un microscope à dissection pour vous assurer qu’il est à plat et non près de bulles d’air.Si le tissu SV n’est pas orienté correctement, essayez d’expulser les bulles d’air ou retirez soigneusement le plus petit couvercle en verre et repositionnez le SV. Une monture entière de SV de souris P30 a été préparée et une imagerie confocale a été réalisée. L’expression de ZsGreen a été observée dans les cellules intermédiaires, les cellules endothéliales marquées par GSIB4 et les noyaux marqués par DAPI.