Dec 20, 2024

Public workspaceImmunohistochemical staining of wholemount female rat urethra preparations

Forked from a private protocol
DOI
dx.doi.org/10.17504/protocols.io.x54v92d64l3e/v1
  • 1University of Melbourne
  • SPARC
    Tech. support email: info@neuinfo.org
John-Paul Fuller-Jackson
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.x54v92d64l3e/v1
Protocol Citation: John-Paul Fuller-Jackson, Peregrine B Osborne, Janet R Keast 2024. Immunohistochemical staining of wholemount female rat urethra preparations. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v92d64l3e/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
Created: May 21, 2024
Last Modified: December 20, 2024
Protocol Integer ID: 100170
Keywords: urethra, myelin, sensory, lower urinary tract, peripheral nervous system
Funders Acknowledgements:
NIH SPARC
Grant ID: 3OT2OD023872
Abstract
This protocol describes the tissue preparation and immunohistochemical procedures applied to wholemount urethra from the female rat. Wholemount immunohistochemistry keeps structures within the tissue intact, but keeps the tissue flat enough that high resolution confocal microscopy can be employed. This is only suitable for the female rat, as the male rat urethra is too thick and muscular. The antibodies recommended in this protocol are to visualize AAV-PHP.S transfected neuron axons, as well as myelin basic protein and S100.
Materials
ReagentAnti-CGRP antibodyAbcamCatalog #AB81887 ReagentHorse serumSigma AldrichCatalog #12449C ReagentTriton X-100Sigma AldrichCatalog #T8787-50ML
ReagentAnti-S100 antibody (rabbit)DakoCatalog #Z0311
ReagentAnti-CGRP antibody (mouse)AbcamCatalog #AB81887
ReagentAnti-MBP antibody (human) conjugated to FITCMiltenyi BiotecCatalog #130-120-341 ReagentAnti-RFP antibody (guinea pig)Synaptic SystemsCatalog #390004
ReagentAnti-TH antibody (rabbit)Merck Millipore (EMD Millipore)Catalog #AB152
ReagentAnti-VAChT antibody (rabbit)Synaptic SystemsCatalog #139103
ReagentAF647 Donkey anti-rabbit antibodyInvitrogenCatalog #A32795 ReagentCy3 Donkey anti-guinea pig IgGJackson ImmunoResearch Laboratories, Inc.Catalog #706-165-148
ReagentAF488 Donkey anti-mouse IgGJackson ImmunoResearch Laboratories, Inc.Catalog #715-545-150

Solutions:

  • PBS: phosphate-buffered saline, 0.1 M, pH 7.2
  • PBS containing 0.1% sodium azide
  • PB: phosphate-buffer, 0.1M, pH7.2
  • Blocking solution: PBS containing 10% normal horse serum and 0.5% triton X-100
  • PBS containing 0.1% sodium azide, 2% normal horse serum and 0.5% triton X-100
  • 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4
  • 0.9% sodium chloride, 1% sodium nitrite (w/v) and 0.11 ml heparin (5000 IU/ml)

Primary Antibodies:


ABCDE
AbbreviationSynonymRRIDHost SpeciesDilution
S100S100AB_10013383Rabbit1:1000
MBP-FITCMyelin basic protein conjugated to Fluorescein isothiocyanateAB_2857520Human1:2000
RFPRed fluorescent proteinAB_2737052Guinea pig1:500
TH Tyrosine hydroxylaseAB_390204Rabbit1:2000
CGRPCalcitonin gene-related peptideAB_1658411Mouse1:1000
VAChTVesicular acetylcholine transporterAB2247684Rabbit1:4000


Secondary Antibodies:


Tag-antibodyHost speciesRRIDDilution
Cy3 anti-guinea-pigdonkeyAB_2340460 1:2000
AF647 anti-rabbitdonkeyAB_27628351:1000
AF488 anti-mousedonkeyAB_23408461:2000






Preparation of sample
Preparation of sample
Following 'Intracardiac perfusion with buffer for anatomical studies'' dissect urethra from the female rat and place it in a petri dish containing prewash solution.
Bisect the urethra longitudinally on the dorsal midline.
Pin the urethra out flat on a Sylgard-lined Petri dish with the urothelial surface facing upwards and gently stretch equally on both edges of the tissue by moving the pinned tissue out until the tissue is completely flat. Do this under stereomicroscope to ensure no damage is done to tissue during this procedure.
Replace the prewash solution with freshly made 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) and post-fix overnight at 4 °C
Remove the pins and wash the urethra in PB (3 x 30 min)
Store in PBS containing 0.1% sodium azide at 4 °C until used for immunohistochemistry
Immunohistochemistry
Immunohistochemistry
Wash urethra in phosphate buffer (PB; 0.1M; pH 7.2) (3 x 10 min)
Wash urethra in 50% ethanol/ H2O (2 x 30 min)
Incubate sections in blocking solution (PB; 10% normal horse serum) at room temperature for 2 h
Incubate sections in appropriate dilutions of primary antibodies (or combinations of primary antibodies) for 5 days. Antibodies are diluted in PBS containing 0.1% sodium azide, 10% normal horse serum, and 0.5% Triton-X100.
Wash tissue in PBS (3 x 30 min)
Incubate sections in appropriate dilutions of secondary antibodies (or combinations of secondary antibodies) 5 days in protected from light. Antibodies are diluted in PBS-azide containing 10% horse serum, and 0.5% triton-X.
Wash tissue in PBS (3 x 30 min), protected from light
1 x 1 hour wash in buffered glycerol
Mount the flat urethra on a glass slide using buffered glycerol and coverslip, seal the slide with clear nail polish. For optimal imaging ensure the tissue layer of interest is facing up towards the coverslip. For example, if the lamina propria and urothelium are to be imaged, place the sample with urothelium up.