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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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NameCompanyCatalog NumberComments
10-µm filter (Polyethylenterephthalat)PluriSelect#43-50010-01Filter tissue during sNuc-Seq
18 x 18 mm cover glassFisher Scientific12-541ACover slip to mount SV
30-µm filter (Polyethylenterephthalat)PluriSelect#43-50030-03Filter tissue during sNuc-Seq
75 x 25 mm Superfrost Plus/Colorforst Plus MicroslideDaiggerEF15978ZMicroslide to mount SV on
C57BL/6J MiceThe Jackson LaboratoryRRID: IMSR_JAX:000664General purpose mouse strain that has pigment more easily seen in the intermediate cells of the SV.
Cell CounterLogos BiosystemsL20001Used for cell counting
Chalizon curette 5'', size 3 2.5 mmBiomedical Research Instruments15-1020Used to transfer SV
Chromium Next GEM single Cell 3' GEM Kit v3.1ChromiumPN-1000141Generates single cell 3' gene expression libraries
Clear nail polishFisher ScientificNC1849418Used for sealing SV mount
Corning Falcon Standard Tissue Culture Dishes, 24 wellCorning08-772BCulture dish used to hold specimen during dissection
DAPIInvitrogenD1306, RRID: AB_2629482Stain used for nucleus labeling
Dounce homogenizerSigma-AldrichD8938Used to homogenize tissue for sNuc-seq
Dumont #5 ForcepsFine Science Tools11252-30General forceps for dissection
Dumont #55 ForcepsFine Science Tools11255-20Forceps with fine tip that makes SV manipulation easier
Fetal Bovine SerumThermoFisher16000044Used for steps of sNuc-Seq
Glue stickFisher ScientificNC0691392Used for mounting SV
GS-IB4 AntibodyMolecular ProbesI21411, RRID: AB-2314662Antibody used for capillary labeling
KCNJ10-ZsGreen Micen/an/aTransgenic mouse that expresses KCNJ10-ZsGreen, partiularly in the intermediate cells of the SV.
MgCl2ThermoFisherAM9530GUsed for steps of sNuc-Seq
Mounting reagentThermoFisher#S36940Mounting reagent for SV
Multiwell 24 well plateCorning#353047Plate used for immunostaining
NaClThermoFisherAAJ216183Used for steps of sNuc-Seq
Nonidet P40Sigma-Aldrich9-16-45-9Used for steps of sNuc-Seq
Nuclease free waterThermoFisher4387936Used for steps of sNuc-Seq
Orbital shakerSilent ShakeSYC-2102AUsed for steps of immunostaining
PBSThermoFisherJ61196.APUsed for steps of immunostaining and dissection
RNA LaterInvitrogenAM7021Used for preservation of SV for sNuc-Seq
ScizzorsFine Science Tools14058-09Used for splitting mouse skull
Tris-HClSigma-Aldrich15506017Used for steps of sNuc-Seq
Trypan blue stainGibco15250061Used for cell counting
Tween20ThermoFisherAAJ20605AP Used for steps of sNuc-Seq
Zeiss STEMI SV 11 Apo stereomicroscopeZeissn/aMicroscope used for dissections

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