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

Microdissecção da Estria Vascular para Sequenciamento de Núcleo Único ou Imunomarcação

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 em destaque: Dissecção de estria vascular de camundongo adulto para sequenciamento de núcleo único ou imunocoloração  

Dissecção da estria vascular de camundongos adultos para sequenciamento de núcleo único ou imunomarcação

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  

Imunomarcação da estria vascular de camundongos e montagem tecidual

Prepare-se para a imunomarcação dissecando a estria vascular do camundongo e adicione o tecido a uma placa de 24 poços contendo 200 microlitros de paraformaldeído a 4% em PBS 1X. Incubar o tecido durante 20 minutos à temperatura ambiente. realizar duas lavagens curtas de 1X PBS em um agitador orbital a baixas RPM.Remova o PBS e, em seguida, realize a permeabilização e o bloqueio por um período mínimo de uma hora à temperatura ambiente em 300 microlitros de solução de PBST. Em seguida, manchar o tecido com o anticorpo primário durante a noite a quatro graus Celsius. Em seguida, adicione um anticorpo secundário e incube por duas horas à temperatura ambiente em um agitador orbital a baixas RPM.Cubra a placa para proteger o anticorpo secundário marcado fluorescentemente da luz. Em seguida, prepare uma microlâmina maior colocando duas pequenas faixas de cola ao longo do eixo de 25 mm, um pouco menor do que a tampa de vidro de 18 por 18 milímetros. Coloque uma gota de reagente de montagem entre as estrias de cola.Usando pinça número 55, coloque o mínimo de tecido possível em uma das extremidades da VS e transfira-o do PBS para o reagente de montagem. Coloque uma extremidade da tampa deslizante na lâmina onde foi colocada uma faixa de cola. Em seguida, solte suavemente a tampa para evitar a criação de bolhas de ar.Sele o suporte passando uma gota de esmalte transparente em cada canto do suporte. Rotule o espécime na tampa de vidro, longe do campo de visualização. Visualize o tecido SV sob um microscópio de dissecção para garantir que ele esteja deitado e não perto de bolhas de ar.Se o tecido do SV não estiver orientado corretamente, tente empurrar as bolhas de ar ou remova cuidadosamente a tampa de vidro menor e reposicione o SV. Uma montagem inteira de VS de camundongo P30 foi preparada e imagens confocais foram realizadas. A expressão de ZsGreen foi observada nas células intermediárias, as células endoteliais marcadas com GSIB4 e os núcleos marcados com DAPI.