Apr 23, 2020

Public workspaceImmunohistochemical labelling of spinal cord neurons involved in bladder activity

DOI
dx.doi.org/10.17504/protocols.io.bakkicuw
  • 1University of Melbourne
  • SPARC
    Tech. support email: info@neuinfo.org
Janet R Keast
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.bakkicuw
Protocol Citation: Janet R Keast, Peregrine B Osborne, Nicole Wiedmann 2020. Immunohistochemical labelling of spinal cord neurons involved in bladder activity. protocols.io https://dx.doi.org/10.17504/protocols.io.bakkicuw
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: December 17, 2019
Last Modified: November 27, 2023
Protocol Integer ID: 31084
Keywords: activity-mapping, neuroanatomy, immunohistochemistry
Abstract
This protocol is used for immunohistochemical visualisation of immediate early gene expression (c-Fos or Egr-1) in cryosections of rat lumbosacral spinal cord. Free-floating sections are processed in a double labelling protocol to distinguish immediate early gene expression in different neurochemical classes of spinal cord neurons:

  • ChAT [choline acetyltransferase]: preganglionic neurons
  • TH [tyrosine hydroxyls]: dopaminergic neurons
  • Pax2: inhibitory interneurons
Materials
MATERIALS
ReagentHorse serumMerck MilliporeSigma (Sigma-Aldrich)Catalog #12449C
ReagentOCT (Optimal Cutting Temperature compound)Sakura FinetekCatalog #4583
ReagentCy3 Donkey anti-goat IgGJackson ImmunoResearch Laboratories, Inc.Catalog #705-165-147
ReagentAF488 Donkey anti-rabbit IgGJackson ImmunoResearch Laboratories, Inc.Catalog #711-545-152
ReagentNeuroTrace™ 640/660 Deep-Red Fluorescent Nissl Stain - Solution in DMSOThermo FisherCatalog #N21483
ReagentMouse anti-cFos antibody Santa Cruz BiotechnologyCatalog #sc166940
ReagentRabbit anti-Pax2 antibodyInvitrogen - Thermo FisherCatalog #71-6000
ReagentRabbit anti-Egr-1 (588) antibodySanta Cruz BiotechnologyCatalog #sc-110
ReagentRabbit anti-TH antibodyMerck Millipore (EMD Millipore)Catalog #AB152
ReagentCy3 Donkey anti-mouse IgGJackson ImmunoResearch Laboratories, Inc.Catalog #715-165-150
ReagentGoat anti-ChAT antibodyMerck Millipore (EMD Millipore)Catalog #AB144P
Solutions:

  • PBS: phosphate-buffered saline, 0.1 M, pH 7,2
  • PBS containing 0.1% sodium azide
  • PBS containing 30% sucrose (w/v)
  • 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

Primary Antibodies:


AbbreviationGene nameSynonymRRIDHost SpeciesDilution
cFosFoscFosAB_10609634Mouse1:100
ChATChatCholine acetyltransferaseAB_2079751Goat1:500
Egr-1/ Zif268Egr1/ Zif268Early growth receptor 1AB_2097174Rabbit1:5000
Pax2Pax2Paired box gene 2AB_2533990Rabbit1:1000
THThTyrosine hydroxylaseAB_390204Rabbit1:2000

Secondary Antibodies:

Tag-antibodyHost speciesDilution
Cy3 anti-mouseDonkey1:2000
AF488 anti-rabbitDonkey1:1000
Cy3 anti-goatDonkey1:1000

Preparation of cryosections
Preparation of cryosections
Cryoprotect fixed tissue (L5-S2 spinal cord) in phosphate-buffered saline (PBS; 0.1 M, pH7.2) containing 30% sucrose. This should be performed at 4C, 24-72h prior to cutting.
Embed tissue in cryomold using OCT, freeze in cryostat and cut sections (40 µm), collecting sections progressively across sets of 4 wells to collect 160 µm spaced series.
Immunostaining
Immunostaining
Wash sections in PBS (3 x 10 min)
Incubate sections in blocking solution at room temperature for 2 h
Incubate sections in appropriate dilutions of primary antibodies (or combinations of primary antibodies) for 48-72h. Antibodies are diluted in PBS containing 0.1% sodium azide, 2% horse serum, and 0.5% triton-X.
Wash sections in PBS (3 x 10 min)
Incubate sections in appropriate dilutions of secondary antibodies (or combinations of secondary antibodies) 4 h in the dark. Antibodies are diluted in PBS containing 2% horse serum, and 0.5% triton-X.

Note
A useful counterstain to visualise spinal regions can be included here, by adding NeuroTrace (fluorescent Nissl stain; 1:100) to the secondary antibody incubation.

Wash sections in PBS (3 x 10 min)
Mount sections onto glass slides and coverslip in preferred anti-fade mountant.
Microscope
Microscope
Labeled neurons are counted and classified according to their immunoreactivity, including only nucleated neuronal profiles in the analysis.

Note
For digital analysis, tile-scanning of complete spinal cord sections is recommended, ensuring that the order of sections (rostral to caudal) is noted.