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

用于单核测序或免疫染色的血管纹显微切割

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.

作者聚焦： 用于单核测序或免疫染色的成年小鼠血管纹解剖  

解剖成年小鼠血管纹以进行单核测序或免疫染色

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  

小鼠血管纹的免疫染色和组织安装

通过从小鼠解剖血管纹来准备免疫染色，并将组织添加到含有200微升4%多聚甲醛的1X PBS中的24孔板中。将组织在室温下孵育20分钟。在低转速下在轨道振动台上短时间洗涤两次 1X PBS。取出PBS，然后在室温下在300微升PBST溶液中进行透化和封闭至少一小时。接下来，用一抗在 4 摄氏度下对组织染色过夜。然后加入二抗，并在室温下以低RPM在轨道振荡器上孵育两小时。盖上平板以保护荧光标记的二抗免受光照。接下来，沿着 25 mm 轴放置两条小胶条，仅比 18 x 18 毫米的玻璃盖玻片小，准备一个更大的微载玻片。在胶条之间放置一滴封片剂。使用55号镊子，在SV的一端尽可能少地吸动组织，并将其从PBS转移到封片试剂中。将盖玻片的一端放在放置一条胶水条纹的载玻片上。然后轻轻松开盖玻片，以免产生气泡。通过在支架的每个角上涂抹一滴透明的指甲油来密封支架。在玻璃盖玻片上标记标本，远离可视化区域。在解剖显微镜下观察SV组织，以确保其平躺且不靠近任何气泡。如果 SV 组织的方向不正确，请尝试推出气泡或小心地取出较小的玻璃盖玻片并重新定位 SV。制备来自P30小鼠的整个SV支架并进行共聚焦成像。在中间细胞中观察到ZsGreen表达，GSIB4标记的内皮细胞和DAPI标记的细胞核。