Apr 02, 2023
Immunohistochemistry using paraffin embedded tissue
- Michael J Hurley1,2
- 1Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, London, UK;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, Maryland, USA
Protocol Citation: Michael J Hurley 2023. Immunohistochemistry using paraffin embedded tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly72ywlx9/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: February 27, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 77671
Keywords: ASAPCRN, Immunohistochemistry, paraffin wax, immunofluorescence, synuclein by immunofluorescence, immunofluorescence, immunohistochemistry, antibody, peroxidase immunohistochemistry, synuclein, tissue this protocol, optimal dilution of the primary antibody, primary antibody, embedded tissue, other antibody, using paraffin, most antibody, many other antibody, only change necessary for most antibody, antigen, paraffin wax, best type of antigen, peroxidase, paraffin, sections of tissue, tissue
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000420
Abstract
This protocol describes how to stain paraffin wax embedded sections of tissue for alpha-synuclein by immunofluorescence or DAB/peroxidase immunohistochemistry and is based on Hurley et al., 2013 (https://doi.org/10.1093/brain/awt134).
The protocol works for many other antibodies and the only change necessary for most antibodies is determining the optimal dilution of the primary antibody and the best type of antigen-retrieval to use.
Materials
Rabbit anti-alpha-synuclein ab212184 Abcam
Goat anti-rabbit IgG biotin111-065-144-JIR Stratech
Goat anti-rabbit IgG Alexa Fluor™ 488 A27034 Thermo Fisher
Streptavidin POD11089153001
Troubleshooting
Wax embedding
Dissect tissue (fresh or trans-cardially perfused with formalin (10% neutral buffered formaldehyde) or 4% paraformaldehyde in PBS).
Safety information
Formaldehyde is toxic by inhalation
Place tissue in at least 5 volumes of fixative at 4 °C for > 48:00:00 if tissue was perfused or > 96:00:00 if fresh. The tissue can stay in fixative indefinitely. If the tissue was fresh and the solution is turbid with blood, change the fixative after 24 hours.
6d
Embed formalin fixed tissue in wax using an automated tissue processor. It can be done manually if you do not have access to an automated tissue processor. Histo-Clear™ II can be used instead of xylene.
Safety information
Xylene is toxic by inhalation and flammable
Ethanol is toxic inhalation and flammable
Gut tissue
1. 10 % formalin 00:01:00 Room temperature
2. 10 % formalin 00:01:00 Room temperature
3. 70 % ethanol 01:00:00 30 °C
4. 90 % ethanol 01:00:00 30 °C
5. 100 % ethanol 01:00:00 30 °C
6. 100 % ethanol 01:00:00 30 °C
7. 100 % ethanol 01:00:00 30 °C
8. 100 % ethanol 01:20:00 30 °C
9. xylene 01:00:00 30 °C
10. xylene 01:00:00 30 °C
11. xylene 01:20:00 30 °C
12. paraffin wax 01:20:00 62 °C
13. paraffin wax 01:20:00 62 °C
14. paraffin wax 01:20:00 62 °C
13h 42m
Brain tissue (whole mouse)
1. 10 % formalin 00:01:00 Room temperature
2. 10% formalin 00:01:00 Room temperature
3. 70% ethanol 02:00:00 30 °C
4. 90% ethanol 02:00:00 30 °C
5. 100% ethanol 02:00:00 30 °C
6. 100% ethanol 02:00:00 30 °C
7. 100% ethanol 02:00:00 30 °C
8. 100% ethanol 02:00:00 30 °C
9. xylene 02:00:00 30 °C
10. xylene 02:00:00 30 °C
11. xylene 04:00:00 30 °C
12. paraffin wax 02:00:00 62 °C
13. paraffin wax 02:00:00 62 °C
14. paraffin wax 02:00:00 62 °C
1d 2h 2m
Mount the tissue in a block of wax using an embedding station and metal or plastic moulds.
Store at Room temperature
Section the tissue (5 µm to 8 µm )using a microtome.
Store sections at Room temperature
Dewaxing and antigen retrieval
Select slides to be stained and place in a microscope slide rack.
Heat sections 60 °C 00:30:00
30m
Dewax sections in two changes of Histo-Clear II (National Diagnostics).
1. 00:10:00
2. 00:10:00
20m
Rehydrate sections through graded (v/v) ethanol solutions.
1. 100 % 00:05:00
2. 100 % 00:05:00
3. 95 % 00:05:00
4. 80 % 00:05:00
5. 70 % 00:05:00
25m
Wash with PBS 00:05:00
5m
Quench endogenous peroxidase with 0.3 % hydrogen peroxide (v/v) 00:05:00
5m
Wash with PBS 00:05:00
5m
For antigen retrieval heat 100 °C the sections in 15 mM sodium citrate buffer containing 0.1% Tween® 20, pH 6 for 00:25:00 in an 800-watt microwave set on 30 % power.
25m
Cool with running tap water 00:05:00
5m
Wash with PBS 2 x 00:05:00
5m
For immunofluorescence staining go to step 17
For DAB/peroxidase staining go to step 27
Immunofluorescence staining
23h 25m
Draw around the sections with a wax pen.
Block non-specific binding sites with 10 % goat serum in PBS containing 0.005 % Triton™ X-100 and 0.05 % thimerosal 01:30:00
Depending on the size and number of sections 150-250 microliters of solution should be sufficient to cover the sections.
1h 30m
Incubate with anti-alpha-synuclein antibody (1:500) (ab212184) in 10 % goat serum containing 0.005 % Triton™ X-100 and 0.05 % thimerosal (antibody buffer) 20:00:00 Room temperature
20h
Wash with PBS 4 x 00:05:00
5m
incubated sections with Alexa Fluor goat anti-rabbit IgG (1:200) (A27034) 01:30:00
1h 30m
Wash with PBS 2 x 00:05:00
5m
incubate sections with DAPI (1 µg/ml) 00:05:00
5m
Wash with PBS 3 x 00:05:00
5m
Dip slides in water to remove salts
Mount sections using Hydromount™ (National Diagnostics).
DAB/peroxidase staining
43m 15s
Draw around the sections with a wax pen.
Block non-specific binding sites with 10 % goat serum in PBS containing 0.005 % Triton™ X-100 and 0.05 % thimerosal 01:30:00
Depending on the size and number of sections 150-250 microliters of solution should be sufficient to cover the sections.
1h 30m
Incubate with anti-alpha-synuclein antibody (1:500) (ab212184) in 10 % goat serum containing 0.005 % Triton™ X-100 and 0.05 % thimerosal (antibody buffer) 20:00:00 Room temperature
20h
Wash PBS 4 x 00:05:00
5m
Incubate sections in biotinylated goat anti-rabbit IgG (1:250) secondary antibody for 2 hours.
Wash PBS 4 x 00:05:00
5m
Incubate sections with streptavidin-peroxidase conjugate (1:250) for 1 hour.
Wash PBS 4 x 00:05:00
5m
Visualise staining by incubation with PBS containing 0.05 % (w/v) 3,3′-Diaminobenzidine/0.015 % (v/v) H2O2/0.05 % (w/v) nickel ammonium sulphate for up to 00:03:00 ,
3m
Wash sections for > 00:05:00 in running tap water.
5m
Counter stain with haematoxylin 00:00:05 to 00:00:10 if desired.
15s
Wash sections with running tap water until it runs clear to remove excess stain.
Dehydrate sections through graded (v/v) ethanol solutions
70 % 00:05:00
80 % 00:05:00
95 % 00:05:00
100 % 00:05:00
100 % 00:05:00
25m
Clear sections with Histo-Clear II (National Diagnostics) 2 x 00:05:00
5m
Mount with Omnimount™ (National Diagnostics) or equivalent permanent resin.
Protocol references