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の微小解剖による単一核シーケンシングまたは免疫染色

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.

著者スポットライト:単一核シーケンシングまたは免疫染色のための成体マウス血管筋の解剖  

単核シーケンシングまたは免疫染色のための成体マウス脈条血管の解剖

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  

マウス脈条血管の免疫染色と組織マウント

マウスから脈管線条を解剖して免疫染色の準備をし、1X PBS中の200マイクロリットルの4%パラホルムアルデヒドを含む24ウェルプレートに組織を追加します。組織を室温で20分間インキュベートします。低RPMのオービタルシェーカーで1X PBSを2回短時間洗浄します。PBSを除去し、300マイクロリットルのPBST溶液中で室温で最低1時間透過処理とブロッキングを行います。次に、一次抗体で組織を4°Cで一晩染色します。次に、二次抗体を添加し、低RPMのオービタルシェーカー上で室温で2時間インキュベートします。プレートを覆い、蛍光タグ付きの二次抗体を光から保護します。次に、18 x 18 mmのガラスカバースリップよりも小さい25 mmの軸に沿って2本の小さな接着剤の縞を配置して、より大きなマイクロスライドを準備します。接着剤の縞の間に1滴の取り付け試薬を置きます。55番の鉗子を使用して、SVの一端の組織をできるだけ少なくし、PBSから封入試薬に移します。接着剤の1本の縞が置かれたスライドにカバースリップの一方の端を置きます。次に、気泡が発生しないようにカバースリップをそっと放します。マウントの各コーナーに透明なマニキュアを一滴軽くたたき、マウントを密封します。可視化フィールドから離れたガラスカバースリップに試料にラベルを付けます。SV組織を解剖顕微鏡で視覚化し、気泡の近くに平らに横たわっていないことを確認します。SV組織の向きが正しくない場合は、気泡を押し出すか、小さいガラスカバースリップを慎重に取り外してSVの位置を変えてみてください。P30マウスからのSVの全マウントを調製し、共焦点イメージングを行った。ZsGreen発現は、中間細胞、GSIB4標識内皮細胞、およびDAPI標識核において観察された。