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

Single-nucleus sequencing 또는 immunostaining을 위한 Stria Vascularis 미세절개

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.

저자 스포트라이트: Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining (단핵 염기서열분석 또는 면역염색을 위한 성체 마우스 줄무늬 혈관 절제)  

단일 핵 시퀀싱 또는 면역염색을 위한 성인 마우스 줄무늬 혈관의 해부

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  

마우스 선조체 혈관의 면역염색 및 조직 장착

마우스에서 선조체 혈관을 해부하여 면역염색을 준비하고 1X PBS에 200마이크로리터의 4% 파라포름알데히드가 들어 있는 24웰 플레이트에 조직을 추가합니다. 실온에서 20분 동안 조직을 배양한다. 낮은 RPM에서 오비탈 셰이커에서 1X PBS를 두 번 짧게 세척합니다.PBS를 제거하고, 300 마이크로리터의 PBST 용액에서 실온에서 최소 1시간 동안 투과화 및 블로킹을 수행하였다. 다음으로, 섭씨 4도에서 밤새 1 차 항체로 조직을 염색합니다. 그런 다음 2차 항체를 첨가하고 낮은 RPM의 오비탈 셰이커 상에서 실온에서 2시간 동안 인큐베이션합니다.형광 태그된 2차 항체를 빛으로부터 보호하기 위해 플레이트를 덮습니다. 그런 다음 25mm 축을 따라 18 x 18mm 유리 커버 슬립보다 작은 두 개의 작은 접착제 줄무늬를 배치하여 더 큰 마이크로 슬라이드를 준비합니다. 접착제 줄무늬 사이에 장착 시약 한 방울을 놓습니다.55번 집게를 사용하여 SV의 한쪽 끝에 가능한 한 적은 조직을 갭으로 밟고 PBS에서 장착 시약으로 옮깁니다. 덮개 슬립의 한쪽 끝을 접착제 한 줄무늬가 놓인 슬라이드에 놓습니다. 그런 다음 기포가 생기지 않도록 덮개 슬립을 부드럽게 풉니다.마운트의 각 모서리에 투명 매니큐어 한 방울을 두드려 마운트를 밀봉합니다. 유리 덮개 슬립에 표본을 시각화 필드에서 멀리 표시하십시오. SV 조직을 해부 현미경으로 시각화하여 기포 근처에 있지 않고 평평하게 놓여 있는지 확인합니다.SV 조직의 방향이 올바르지 않으면 기포를 밀어내거나 작은 유리 커버 슬립을 조심스럽게 제거하고 SV의 위치를 조정하십시오. P30 마우스로부터 SV의 전체 마운트를 준비하고, 공초점 이미징을 수행하였다. ZsGreen 발현은 중간 세포, GSIB4 표지된 내피 세포, 및 DAPI 표지된 핵에서 관찰되었다.