Aug 15, 2024
Immunohistochemical staining of wholemount major pelvic ganglia (MPG) for analysis of myelinated bladder afferents
Forked from a private protocol
- 1University of Melbourne
- SPARCTech. support email: info@neuinfo.org
Protocol Citation: Janet R Keast, Peregrine B Osborne, Nicole Wiedmann 2024. Immunohistochemical staining of wholemount major pelvic ganglia (MPG) for analysis of myelinated bladder afferents. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvme3nbg3p/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: May 23, 2024
Last Modified: August 15, 2024
Protocol Integer ID: 100336
Funders Acknowledgements:
NIH SPARC
Grant ID: 3OT2OD023872
Abstract
This protocol describes immunohistochemical procedures applied to wholemount major pelvic ganglia (MPG) for the visualization of myelinated axons filled with cholera toxin subunit B (CTB) and/or neurons transfected with adeno-associated virus (AAV) expressing TdTomato. In this protocol, samples were obtained from rats in which CTB was microinjected into the bladder, and AAV-PHP.S was intravenously administered to preferentially and sparsely label peripheral neurons. In this context, neurofascin (paranode marker) identifies myelinated axons.
Materials
Horse serumSigma AldrichCatalog #12449C Triton X-100Sigma AldrichCatalog #T8787-50ML Anti-RFP antibody (guinea pig)Synaptic SystemsCatalog #390004 Anti-cholera toxin subunit B antibody (goat)List LabsCatalog #703
Anti-neurofascin antibody (rabbit)Alomone LabsCatalog #AIP-025
Cy3 Donkey anti-guinea pig IgGJackson ImmunoResearch Laboratories, Inc.Catalog #706-165-148
AF488 Donkey anti-goat IgGJackson ImmunoResearch Laboratories, Inc.Catalog #705-545-147
AF647 Donkey anti-rabbit antibodyInvitrogenCatalog #A32795
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
Primary Antibodies:
| A | B | C | D | E | |
| Abbreviation | Synonym | RRID | Host Species | Dilution | |
| RFP | Red fluorescent protein | AB_2737052 | Guinea pig | 1:500 | |
| CTB | Cholera toxin subunit B | AB_10013220 | Goat | 1:10,000 | |
| Neurofascin | NF155 | AB_2756657 | Rabbit | 1:1000 |
Secondary Antibodies:
| A | B | C | D | |
| Tag-antibody | Host Species | RRID | Dilution | |
| Cy3 anti-guinea pig | Donkey | AB_2340460 | 1:2000 | |
| AF488 anti-goat | Donkey | AB_2336933 | 1:1000 | |
| AF647 anti-rabbit | Donkey | AB_2762835 | 1:1000 |
Immunohistochemistry
Immunohistochemistry
Wash whole MPGs in phosphate buffer (PB; 0.1M; pH 7.2) (3 x 30 min)
Incubate sections in blocking solution (PB; 10% horse serum; and 0.5% Triton-X) at room temperature for 2 h
Incubate sections in appropriate dilutions of primary antibodies (or combinations of primary antibodies) for 72h. Antibodies are diluted in PBS containing 0.1% sodium azide, 2% horse serum, and 0.5% triton-X.
Wash tissue in PBS (3 x 30 min)
Incubate sections in appropriate dilutions of secondary antibodies (or combinations of secondary antibodies) 24 h in the dark. Antibodies are diluted in PBS containing 2% horse serum, and 0.5% triton-X.
Wash tissue in PBS (3 x 30 min)
Mount tissue onto glass slides and coverslip in preferred anti-fade mountant.