Apr 23, 2020
Immunohistochemical labeling of thick cryosections from pelvic ganglia
- 1University of Melbourne
- SPARCTech. support email: info@neuinfo.org
Protocol Citation: Janet R Keast, Peregrine B Osborne 2020. Immunohistochemical labeling of thick cryosections from pelvic ganglia. protocols.io https://dx.doi.org/10.17504/protocols.io.bakcicsw
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: 31076
Keywords: immunohistochemistry; immunofluorescence
Abstract
This protocol describes immunohistochemical procedures applied to thick (50 µm) cryosections mounted directly on slides. It is used when the structures to be analysed are too large to remain intact within thin (10-20 µm) cryosections. Antibodies have been selected to distinguish different neurochemical classes of autonomic ganglion neurons and synaptic boutons associated with these neurons. The protocol can also be used to characterize neurons containing retrograde tracer.
Materials
MATERIALS
Horse serumMerck MilliporeSigma (Sigma-Aldrich)Catalog #12449C
OCT (Optimal Cutting Temperature compound)Sakura FinetekCatalog #4583
Sheep anti-neuronal nitric oxide synthase antibody; AB_90743Merck Millipore (EMD Millipore)Catalog #AB1529
Cy3 Donkey anti-goat IgGJackson ImmunoResearch Laboratories, Inc.Catalog #705-165-147
AF488 Donkey anti-rabbit IgGJackson ImmunoResearch Laboratories, Inc.Catalog #711-545-152
PAP hydrophobic barrier penEnzo Life SciencesCatalog #ADI-950-233-0001
Triton X-100Merck Millipore (EMD Millipore)Catalog #X100
Vectorshield anti-fade mounting mediumVector LaboratoriesCatalog #H-1000
Mouse anti-synaptophysin antibodyAgilent TechnologiesCatalog #M0776
Rabbit anti-protein gene product 9.5 antibodyMerck Millipore (EMD Millipore)Catalog #B5925
Sheep anti-tyrosine hydroxylase antibodyMerck Millipore (EMD Millipore)Catalog #AB1542
AF647 Donkey anti-mouse IgGThermofisherCatalog #A-31571
Solutions:
- PBS: phosphate-buffered saline, 0.1 M, pH 7,2
- PBS containing 30% sucrose (w/v)
- PBS containing 0.1% sodium azide
- Blocking solution: PBS containing 10% non-immune horse serum and 0.5% triton X-100
Primary antibodies:
| Abbreviation | Gene name | Synonym | RRID | Host species | Dilution | |
| nNOS | Nos1 | Neuronal nitric oxide synthase | AB_90743 | sheep | 1:2000 | |
| PGP | Uchl1 | Protein gene product 9.5; ubiquitin C-terminal hydrolase 1 | AB_11214054 | rabbit | 1:2000 | |
| SYP | Syp | Synaptophysin | AB_2199013 | mouse | 1:300 | |
| TH | Th | Tyrosine hydroxylase | AB_90755 | sheep | 1:1000 |
Neuronal cell bodies are labelled with PGP, nNOS or TH antibodies. Synaptic boutons associated with these cells are identified with synaptophysin-immunoreactivity.
Secondary antibodies:
| Tag-antibody | Host species | Dilution | |
| AF488 anti-rabbit | Donkey | 1:1000 | |
| AF647 anti-mouse | Donkey | 1:500 | |
| Cy3 anti-goat | Donkey | 1:1000 |
Preparation of cryosections
Preparation of cryosections
Cryoprotect fixed tissue 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 (50 µm), distributing sections progressively across sets of 3-4 slides so that each slide has non-consecutive sections.
Note
Chrome-alum treated slides are pre-coated (subbed) with 1% gelatin, dried and stored at room temperature. This slide treatment enhances section adhesion while retaining good surface tension of the antibody droplets.
Immunostaining
Immunostaining
Air-dry slides at room temperature for at least 10 minutes.
Draw around sections with hydrophobic barrier pen (PAP pen); wait for 10 min to dry.
Wash sections in PBS (10 min).
Incubate sections in blocking solution (PBS containing 10% non-immune horse serum and 0.5% triton X-100) at room temperature in a humidified dark chamber for 2 h.
Incubate sections in appropriate dilutions of primary antibodies (or combinations of primary antibodies) for 48h in a humidified dark chamber. Antibodies are diluted in PBS containing 2% horse serum, 0.5% Triton and 0.1% sodium azide.
Wash sections in PBS (30 min)
Incubate sections in appropriate dilutions of secondary antibodies (or combinations of secondary antibodies) for 4h in a humidified dark chamber. Antibodies are diluted in PBS containing 2% horse serum, 0.5% Triton and 0.1% sodium azide.
Wash sections in PBS (30 min)
Coverslip in Vectorshield or preferred anti-fade mountant.