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Biology

Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining

Published: April 21st, 2023

DOI:

10.3791/65254

1Auditory Development and Restoration Program, National Institute on Deafness and Other Communication Disorders, National Institutes of Health

The stria vascularis is vital to the generation of endocochlear potential. Here, we present the dissection of the adult mouse stria vascularis for single-nucleus sequencing or immunostaining.

Endocochlear potential, which is generated by the stria vascularis, is essential to maintain an environment conducive to appropriate hair cell mechanotransduction and ultimately hearing. Pathologies of the stria vascularis can result in a decreased hearing. Dissection of the adult stria vascularis allows for focused single-nucleus capture and subsequent single-nucleus sequencing and immunostaining. These techniques are used to study stria vascularis pathophysiology at the single-cell level.

Single-nucleus sequencing can be used in the setting of transcriptional analysis of the stria vascularis. Meanwhile, immunostaining continues to be useful in identifying specific populations of cells. Both methods require proper stria vascularis dissection as a prerequisite, which can prove to be technically challenging.

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 ....

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All animal experiments and procedures were performed according to protocols approved by the Animal Care and Use Committee of the National Institute of Neurological Diseases and Stroke and the National Institute on Deafness and Other Communication Disorders, National Institutes of Health. All experimental protocols were approved by the Animal Care and Use Committee of the National Institute of Neurological Diseases and Stroke and the National Institute on Deafness and Other Communication Disorders, National Institutes of .......

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We present a method to isolate the SV to be used for either sNuc-Seq or immunostaining. The relevant anatomy (Figure 1) of the cochlea relative to the SV can help users better understand the organization of the SV and steps of the dissection protocol.

Each step of this microdissection of SV from a P30 mouse is detailed in the associated video, and snapshots of the key steps of this dissection and isolation of SV are presented in Figure 2

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Prior to the advent of single-cell sequencing, many researchers used bulk tissue analysis, which only made it possible to analyze transcriptomes averaged across cells. In particular, single-cell and sNuc-Seq made it possible to isolate the transcriptome of a single cell or single nucleus, respectively32. In this instance, single-nucleus transcriptomes can be identified for marginal, intermediate, and basal cells, as well as spindle cells30. This enables the investigation of.......

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This research was supported in part by the Intramural Research Program of the NIH, NIDCD to M.H. (DC000088)

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Name Company Catalog Number Comments
10-µm filter (Polyethylenterephthalat) PluriSelect #43-50010-01 Filter tissue during sNuc-Seq
18 x 18 mm cover glass Fisher Scientific 12-541A Cover slip to mount SV
30-µm filter (Polyethylenterephthalat) PluriSelect #43-50030-03 Filter tissue during sNuc-Seq
75 x 25 mm Superfrost Plus/Colorforst Plus Microslide Daigger EF15978Z Microslide to mount SV on
C57BL/6J Mice The Jackson Laboratory RRID: IMSR_JAX:000664 General purpose mouse strain that has pigment more easily seen in the intermediate cells of the SV.
Cell Counter Logos Biosystems L20001 Used for cell counting
Chalizon curette 5'', size 3 2.5 mm Biomedical Research Instruments 15-1020 Used to transfer SV
Chromium Next GEM single Cell 3' GEM Kit v3.1 Chromium PN-1000141 Generates single cell 3' gene expression libraries
Clear nail polish Fisher Scientific NC1849418 Used for sealing SV mount
Corning Falcon Standard Tissue Culture Dishes, 24 well Corning 08-772B Culture dish used to hold specimen during dissection
DAPI Invitrogen D1306, RRID: AB_2629482 Stain used for nucleus labeling
Dounce homogenizer Sigma-Aldrich D8938 Used to homogenize tissue for sNuc-seq
Dumont #5 Forceps Fine Science Tools 11252-30 General forceps for dissection
Dumont #55 Forceps Fine Science Tools 11255-20 Forceps with fine tip that makes SV manipulation easier
Fetal Bovine Serum ThermoFisher 16000044 Used for steps of sNuc-Seq
Glue stick Fisher Scientific NC0691392 Used for mounting SV
GS-IB4 Antibody Molecular Probes I21411, RRID: AB-2314662 Antibody used for capillary labeling
KCNJ10-ZsGreen Mice n/a n/a Transgenic mouse that expresses KCNJ10-ZsGreen, partiularly in the intermediate cells of the SV.
MgCl2 ThermoFisher AM9530G Used for steps of sNuc-Seq
Mounting reagent ThermoFisher #S36940 Mounting reagent for SV
Multiwell 24 well plate Corning #353047 Plate used for immunostaining
NaCl ThermoFisher AAJ216183 Used for steps of sNuc-Seq
Nonidet P40 Sigma-Aldrich 9-16-45-9 Used for steps of sNuc-Seq
Nuclease free water ThermoFisher 4387936 Used for steps of sNuc-Seq
Orbital shaker Silent Shake SYC-2102A Used for steps of immunostaining
PBS ThermoFisher J61196.AP Used for steps of immunostaining and dissection
RNA Later Invitrogen AM7021 Used for preservation of SV for sNuc-Seq
Scizzors Fine Science Tools 14058-09 Used for splitting mouse skull
Tris-HCl Sigma-Aldrich 15506017 Used for steps of sNuc-Seq
Trypan blue stain Gibco 15250061 Used for cell counting
Tween20 ThermoFisher AAJ20605AP  Used for steps of sNuc-Seq
Zeiss STEMI SV 11 Apo stereomicroscope Zeiss n/a Microscope used for dissections

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