Jan 15, 2025
Preparation of Mouse Kidney for Spatial and Single-Cell Transcriptomics
- Shuoshuo Wang1,2,3
- 1Beth Israel Deaconess Medical Center;
- 2Broad Institute of MIT and Harvard;
- 3Harvard Medical School
- protocols.io Ambassadors
Protocol Citation: Shuoshuo Wang 2025. Preparation of Mouse Kidney for Spatial and Single-Cell Transcriptomics. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4wx12vo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 08, 2025
Last Modified: January 15, 2025
Protocol Integer ID: 117865
Keywords: kidney, mouse, spatial, single cell, method, dissection, preclinical
Funders Acknowledgements:
NIAID
Grant ID: P01AI179405
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Abstract
This protocol describes the preparation of murine kidney specimens for single-cell and spatial transcriptomics through a non-survival procedure. The protocol can also be adapted for immunohistochemistry, immunofluorescence, or other molecular assays.
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Materials
10cm dish can be used instead, too.
Corning® non-treated culture dishes 60 mm × 15 mmMerck MilliporeSigma (Sigma-Aldrich)Catalog #CLS430589
Eppendorf Tubes 5.0 mL, screw cap, (Catalog No. 0030122313)
Integra Miltex Sterile Disposable Scalpels, Blade No. 10 (Fisher, Catalog No. 12-460-451)
Protocol materials
Ethyl alcohol, Pure 200 proof, for molecular biology Merck MilliporeSigma (Sigma-Aldrich)Catalog #E7023
Paraformaldehyde Aqueous Solution -16%Electron Microscopy SciencesCatalog #15700
Gibco™ DPBS no calcium no magnesiumThermo Fisher ScientificCatalog #14190144
Gibco™ DPBS no calcium no magnesiumThermo Fisher ScientificCatalog #14190144
Dulbeccos phosphate-buffered saline (DPBS)Gibco - Thermo Fisher ScientificCatalog #14190144
16% ParaformaldehydeElectron Microscopy SciencesCatalog #15710
PBS - Phosphate-Buffered Saline (10X) pH 7.4Thermo Fisher ScientificCatalog #AM9625
Corning® non-treated culture dishes 60 mm × 15 mmMerck MilliporeSigma (Sigma-Aldrich)Catalog #CLS430589
Safety warnings
This protocol involves animal surgical procedures that must be conducted in strict accordance with all applicable laws, regulations, and ethical standards. Failure to adhere to these guidelines may result in legal consequences, including revocation of research privileges, sanctions, or other enforcement actions.
Ethics statement
The animal handling procedures described in this protocol are provided solely as an illustrative example and must not be implemented without proper approval. The sole authoritative source for the execution of animal handling procedures is the protocol approved by the local Institutional Animal Care and Use Committee (IACUC), in strict accordance with applicable laws, regulations, and institutional policies designed to protect animal welfare.
Following this described method is not a substitute for exercising the utmost diligence and care required when handling animals. Researchers are advised to prioritize animal welfare and scientific integrity at every stage of their work. Caution should be exercised to ensure compliance with all relevant ethical and legal standards, and any deviation from approved protocols must be justified and formally authorized by appropriate regulatory bodies.
By proceeding with this protocol, you acknowledge and agree to adhere to all applicable legal and ethical requirements and to accept responsibility for ensuring the humane and lawful treatment of animals throughout the course of these procedures.
Before start
Prepare all required sterile surgical instruments, including scissors, forceps, and a scalpel.
Razor blades may be used as an alternative to a scalpel, provided they are sterilized.
Precool Dulbeccos phosphate-buffered saline (DPBS)Gibco - Thermo FischerCatalog #14190144
For FFPE fixation, dilute EM-grade
16% ParaformaldehydeElectron Microscopy SciencesCatalog #15710 from a new ampoule in PBS - Phosphate-Buffered Saline (10X) pH 7.4Thermo Fisher ScientificCatalog #AM9625 to make fresh 4% PFA solution shortly before the process.
Animal handling as non-survival surgical procedures
Animal handling as non-survival surgical procedures
Euthanize the mouse using an approved method to ensure minimal distress, adhering to existing IACUC protocols.
A surgical plane of anesthesia must be established before starting the procedure and consistently maintained throughout the surgery until euthanasia is performed.
Safety information
Warning: The procedures outlined in this protocol are examples and should not be applied without approval from your local IACUC and adherence to relevant legal and institutional regulations protecting animal welfare. The exact method used must align with the specific disease model and biological questions being addressed. Improper application may compromise animal welfare and confound experimental outcomes. Maximal caution and due diligence are essential to ensure scientific integrity and ethical compliance.
[Example] Anesthetize mice using
- CO₂ asphyxiation (per AVMA guidelines with appropriate flow rates) or
- inhalation of <5% isoflurane in a properly ventilated hood.
Confirm successful induction of anesthesia before proceeding by checking for the absence of reflexes (e.g., toe pinch test).
[Example] Apply confirmatory cervical dislocation. Secure the head with one hand and the hindquarters or tail with the other. Perform a swift and firm motion to separate the cervical vertebrae.
Confirm death by checking for absence of reflexes and respiration.
Immediately proceed to the tissue collection steps.
Harvesting and grossing
Harvesting and grossing
Place the mouse in the supine position on a sterile surgical pad.
Using sterile dissecting scissors, create a midline incision along the linea alba, starting from the pubis and extending cranially toward the xiphoid process.
Reflect the skin laterally to expose the abdominal wall (musculature and fascia).
Incise the abdominal musculature and peritoneum along the same midline axis, ensuring clean entry into the peritoneal cavity. Avoid damaging underlying viscera.
Gently retract the intestines laterally using sterile forceps or moistened gauze to expose the kidneys in their retroperitoneal location.
Identify the renal fascia and dissect it away using fine forceps or iris scissors to isolate the kidney.
Excise each kidney promptly to minimize warm ischemic time (WIT) by transecting the renal pedicle, including the renal artery, renal vein, and ureter using microsurgical scissors.
Gently recover the kidney individually, minimizing mechanical damage to the renal capsule.
Immediately immerse the kidney in an appropriate medium, such as RNAse-free ice-cold phosphate-buffered saline (dPBS). Gibco™ DPBS no calcium no magnesiumThermo Fisher ScientificCatalog #14190144
Using fine forceps, carefully dissect and remove the perinephric fat in a tissue culture dish.
Using fine surgical scissors, excise a portion of the renal pelvis at its base to eliminate remnants of the ureter.
Superficially pierce the fibrous renal capsule using a fine needle or scalpel blade.
Grasp the capsule at the opening with fine forceps and gently peel it away from the kidney surface, taking care to avoid damaging the underlying renal parenchyma.
Bisect each kidney along the sagittal plane using a sterile, sharp scalpel to ensure clean and precise cuts.
Note
Alternatively, consider using a commercial stainless block or 3D-printed resin block.
Example of a matrix block with a 0.5 mm channel
Keep one half for FFPE and the other half for FF. Or further divide into quadrants for different types of assays and preservation.
Rinse the sample again in RNase-free Gibco™ DPBS no calcium no magnesiumThermo Fisher ScientificCatalog #14190144
to remove blood.
Immediately proceed to fixation and freezing.
Fixation, processing and embedding (FFPE-option)
Fixation, processing and embedding (FFPE-option)
3d 2h 15m
3d 2h 15m
Fix the kidney halves in fresh methanol-free EM-grade 4 % (v/v) paraformaldehyde (PFA) at 4 °C Overnight in a RNase-free screw cap 5 mL tube, ensuring the tissue is fully submerged. Gently shake on an orbital shaker.
Note
Always prepare fresh PFA from methanol free EM-grade PFA out of a new ampoule.
Paraformaldehyde Aqueous Solution -16%Electron Microscopy SciencesCatalog #15700
Unused PFA can be stored shaded from light at 4 °C for no more than 72:00:00 .
In the next morning, remove the fixative and replenish the tube with fresh 4 % (v/v) paraformaldehyde (PFA).
If histologist cannot immediately process the sample, dehydrate the samples in an ascending ethanol series: Ethyl alcohol, Pure 200 proof, for molecular biology Merck MilliporeSigma (Sigma-Aldrich)Catalog #E7023
70 % (v/v) ethanol 00:15:00
15m
90 % (v/v) ethanol 00:15:00
15m
100 % (v/v) ethanol 00:15:00
15m
100 % (v/v) ethanol 00:15:00
15m
100 % (v/v) ethanol 00:30:00
30m
100 % (v/v) ethanol 00:45:00
45m
store them in 100 % (v/v) ethanol before submission to histology for further processing and embedding. The storage time should be limited to less than 72:00:00 .
3d