Sep 16, 2025
Protocol for Investigating Periglomerular Afferent Innervation in Mouse Renal Cortex
- Roman Tyshynsky1,
- Lucy Vulchanova1,
- John Osborn1
- 1University of Minnesota
- SPARCTech. support email: [email protected]
Protocol Citation: Roman Tyshynsky, Lucy Vulchanova, John Osborn 2025. Protocol for Investigating Periglomerular Afferent Innervation in Mouse Renal Cortex. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbwz51gpk/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: August 25, 2025
Last Modified: September 16, 2025
Protocol Integer ID: 225481
Keywords: renal, afferents, glomeruli, juxtamedullary, renal cortex, interoception, potential mechanosensory role of periglomerular afferent axon, periglomerular afferent innervation in mouse renal cortex, afferent axons in the mouse kidney, periglomerular afferent axon, investigating periglomerular afferent innervation, mouse renal cortex, anatomical relationship between renal glomeruli, afferent axon, glomerular pressure, mouse kidney, renal glomeruli, monitoring glomerular pressure, potential mechanosensory role, cgrp
Funders Acknowledgements:
NIH SPARC
Grant ID: U01 DK116320
Abstract
This protocol aims to investigate the anatomical relationship between renal glomeruli and afferent axons in the mouse kidney, focusing on those identified by TRPV1 lineage and CGRP immunolabeling. The study seeks to elucidate the potential mechanosensory role of periglomerular afferent axons in monitoring glomerular pressure.
Materials
Materials -
C57BL/6J Mice
Phosphate Buffered Saline (PBS)
Paraformaldehyde (PFA)
Triton-X100
Bovine Serum Albumin (BSA)
Normal Donkey Serum
CUBIC-L and CUBIC-R+
Gelatin-coated slides
Xylenes
DPX Mountant
Reagents -
Paraformaldehyde
PBS with Triton-X100, BSA, and Donkey Serum
Primary Antibodies -
| A | B | C | D | E | F | G | |
| Name | Target | Host | Vendor | Catalog number | RRID | Dilution | |
| Living Colors DsRed Polyclonal Antibody | DsRed | rabbit | Takara Bio | 632496 | RRID:AB_10013483 | 1:500 | |
| Mouse Nephrin Antibody | Nephrin - mouse | goat | R and D Systems | AF3159 | RRID:AB_2155023 | 1:500 | |
| Sheep Anti-Tyrosine Hydroxylase (TH, Tyrosine Monooxygenase) Polyclonal antibody, Unconjugated | Tyrosine Hydroxylase | sheep | Millipore | AB1542 | RRID:AB_90755 | 1:500 | |
| Anti-Tyrosine Hydroxylase Antibody | Tyrosine Hydroxylase | rabbit | Millipore | AB152 | RRID:AB_390204 | 1:500 | |
| CGRP (Calcitonin Gene Related Peptide) Antibody | rat alpha-CGRP | rabbit | ImmunoStar | 24112 | RRID:AB_572217 | 1:500 | |
Primary antibodies
rabbit anti-DsRed, Takara Bio, San Jose, CA, USA, cat# 632496
goat anti-nephrin, R&D Systems, Minneapolis, MN, USA, cat# AF3159
sheep anti-TH, Millipore, Burlington, MA, USA, cat# AB1542
rabbit anti-TH, Millipore, Burlington, MA, USA, cat# AB152
rabbit anti-CGRP, Immunostar, Hudson, WI, USA, cat#24112
Secondary Antibodies -
| A | B | C | D | E | F | H | |
| Name | Target | Host | Vendor | Catalog number | RRID | Dilution | |
| Cy3-AffiniPure Donkey Anti-Rabbit IgG (H+L) | rabbit IgG (H+L) | donkey | Jackson ImmunoResearch Labs | 711-165-152 | RRID:AB_2307443 | 1:300 | |
| Cy3-AffiniPure Donkey Anti-Sheep IgG (H+L) | sheep IgG (H+L) | donkey | Jackson ImmunoResearch Labs | 713-165-147 | RRID:AB_2315778 | 1:300 | |
| Alexa Fluor 488-AffiniPure Donkey Anti-Goat IgG (H+L) | goat IgG | donkey | Jackson ImmunoResearch Labs | 705-545-147 | RRID:AB_2336933 | 1:300 | |
| Cy5-AffiniPure Donkey Anti-Rabbit IgG (H+L) | rabbit IgG (H+L) | donkey | Jackson ImmunoResearch Labs | 711-175-152 | RRID:AB_2340607 | 1:300 | |
| Cy5-AffiniPure Donkey Anti-Sheep IgG (H+L) | sheep IgG (H+L) | donkey | Jackson ImmunoResearch Labs | 713-175-147 | RRID:AB_2340730 | 1:300 |
Cy3-conjugated donkey anti-rabbit, Jackson ImmunoResearch, West Grove, PA, cat# 711-165-152
Cy3-conjugated donkey anti-sheep, Jackson ImmunoResearch, West Grove, PA, cat# 713-165-147;
Alexa 488-conjugated donkey anti-goat, Jackson ImmunoResearch, West Grove, PA, cat# 705-545-147
Cy5-conjugated donkey anti-rabbit, Jackson ImmunoResearch, West Grove, PA, cat# 711-175-152
Cy5-conjugated donkey anti-sheep, Jackson ImmunoResearch, West Grove, PA, cat# 713-175-147
Troubleshooting
Safety warnings
Handle all reagents and biological materials according to institutional biosafety guidelines. Use personal protective equipment (PPE) including gloves, lab coats, and safety goggles.
Ethics statement
The animal study was reviewed and approved by University of Minnesota Institutional Animal Care and Use Committee.
Anesthetize mice using isoflurane.
Preparation for glomerular scoring analyses15 steps
If renal slices are intended to be used to analyze fiber prevalence near glomeruli en masse, follow this step-case.
Perform cardiac perfusion with ice-cold calcium-free Tyrode’s solution (in mM: NaCl 116, KCl 5.4, MgCl2 1.6, MgSO4 0.4, NaH2PO4 1.4, glucose 5.6, NaHCO3 26) followed by Lana's fixative (4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer 6.9 ).
Tissue Sectioning
Remove kidneys and store in PBS until sectioned.
Decapsulate kidneys and section coronally into 150 µm slices using a Vibratome.
Store serial sections in PBS at 4 °C until staining.
Immunostaining and Xylenes Clearing
4d 0h 30m
Incubate sections in blocking buffer (PBS with 0.3% Triton-X100; 1% BSA, 1% normal donkey serum) at.4 °C Overnight .
1d
Incubate sections in primary antibodies diluted in blocking buffer for 48:00:00 at 4 °C .
2d
Wash sections 3 times for 00:30:00 each in PBS at Room temperature .
30m
Incubate in secondary antibodies for 24:00:00 at Room temperature .
1d
Wash sections again in PBS and mount on gelatin-coated slides.
Dehydrate slide-mounted sections with increasing concentrations of ethanol (50, 75, 100, and 100% in diH2O, 30 minutes each).
Clear slide-mounted sections in Xylenes until tissue appears transparent.
Coverslip using DPX Mountant.
Imaging
Use a Nikon A1R FLIM Confocal Microscope equipped with the A1R GaAsP Confocal system to capture images of immunolabeled sections.
Note
Most slices will be imaged as 5-15 optical sections with a z-step between 5 and 15 µm.
Analysis of whole-slice, low-resolution images ("Glomerular Scoring")
Process composite images into multiple maximum intensity projections of 2-6 optical sections, taking care to ensure that individual glomeruli are not duplicated between projections.
Load each composite image into the custom MATLAB scripts that localizes immunofluorescently labeled glomeruli. Individual glomeruli will be presented for the user to score whether a fine fiber-like structure is closely apposed to the nephrin+ labeling.
Scripts used: Glomerular_Depth_Characterization.m; SmartBorder.m; simpleGlomFinder.m; play_script.m
Acknowledgements
We would like to thank Galina Kalyuzhnaya, Dominic Mussato, and Emma Brown for technical assistance. We would also like to acknowledge the University of Minnesota Imaging Center for its support and expertise throughout the study.