Mar 18, 2024
Immunohistochemistry protocol optimized at iMM-JLA
- 1Comparative Pathology Unit, Instituto de Medicina Molecular - João Lobo Antunes, Lisboa, Portugal
- Ana M Biscaia Santos: Biomedical Scientist Specialist
- Ana M Cristóvão Pinto: Biomedical Scientist
- Ana R Pires: Biomedical Scientist Specialist
- Joana G Antunes: Biomedical Scientist - corresponding author: joana.antunes@medicina.ulisboa.pt
Protocol Citation: Ana M Biscaia Santos, Ana M Cristóvão Pinto, Ana R Pires, Joana G Antunes 2024. Immunohistochemistry protocol optimized at iMM-JLA. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkon2wv5r/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: October 31, 2023
Last Modified: March 18, 2024
Protocol Integer ID: 90197
Keywords: immunohistochemistry, histology, immunostaining, FFPE, frozen, Envision HRP
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Abstract
The immunohistochemistry (IHC) protocol is used to showcase the location of specific proteins in relation to the arquitecture of the tissue.
In this method, we use a primary antibody which binds to a specific antigen in the protein of interest. This antibody has a specific host and can be ready to use or undiluted. The secondary antibody is an anti-host IgG which will bind to the primary antibody 1. It's also conjugated with an Horseradish Peroxidase (HRP).
The HRP will react with the Hydrogen Peroxide in the substract of the DAB solution, oxidizing the DAB, creating a brown deposit on the location of the protein of interest.
Depending on location of the protein, we can have staining in the nuclei, the cytoplasm or the cellular membrane.
Possible variables are:
the concentration of the antibody;
type of antigen retrieval;
the pH of the antigen retrieval solution (for Heat Induced Epitope Retrieval (HIER));
the time of incubation;
the tissue used 2.
You can use this protocol for Formalin-Fixed Paraffin-Embedded samples (FFPE) and Fixed Frozen samples (Gelatin and OCT).
Materials
Coplin Jars
Incubation chamber
Slide Dipper
Slide dishes with Lids
Wash bottles with nozzle
Magnets
Magnetic mixer
Tweezers
Micropipettes p20, p200 and p1000 and respective tips
Hydrophobic PAP pen
Eppendorfs 1.5mL
Plastic pipettes
Equipment Pretreatment Module‱ Deparaffinization and Heat-Induced Epitope Retrieval by Thermo Scientific
Reagents:
Ethanol 70%
Ethanol 96%
Ethanol 100%
Xylene
Distilled Water
Protocol materials
EnVisionTM Flex conjugated w/ HRP DakoCatalog #Ref. K4061
Step 20
Protein Block Serum free DakoCatalog #Ref. X0909
Step 17
EMSURE MethanolSupelcoCatalog #1.06009.2500
In 2 steps
EnvisionTM Dako DAB DakoCatalog #Ref. K3468
Step 22
Harris hematoxylin for histologyBio-opticaCatalog #05-06004E
Step 24
Antibody DiluentDakoCatalog #Ref. 8006
Step 18
EnVision FLEX Target Retrieval Solution High pHDakoCatalog #K8004
Step 10
EnvisionTM Flex Wash Buffer DakoCatalog #Ref. K8007
In 5 steps
Entellan ®Merck MilliporeSigma (Sigma-Aldrich)Catalog #1.00869
Step 29
EnVision FLEX Target Retrieval Solution Low pHDakoCatalog #K8005
Step 10
Hydrogen Peroxide SolutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #7722-84-1
Step 13
Safety warnings
This protocol uses the following hazardous chemicals:
Xylene
Methanol
Hydrogen peroxide
DAB
Resinous Mounting Medium
Please comply with all safety measures and risk assessment protocols.
Latex gloves can be used for the entire protocol, except when manipulating the reagents previously stated, during which you should use nitrile gloves.
Type IV biological disposable bin should be readily available.
Before start
If FFPE - begin protocol after adhering sections to the slides for 1h at 60ºC
Preparation of Wash Buffer
Preparation of Wash Buffer
Measure 20 mL of 20X EnvisionTM Flex Wash Buffer Bio-opticaCatalog #Ref. K8007
Make up with distilled water to 1 L of total volume
Sample Preparation after sectioning - FFPE
Sample Preparation after sectioning - FFPE
Deparaffinize slides with Xylene for 00:10:00
10m
Change Xylene and deparaffinize for another 00:10:00
10m
Place slides in Ethanol 100% for 00:05:00
5m
Place slides in Ethanol 96% for 00:05:00
5m
Place slides in Ethanol 70% for 00:05:00
5m
To complete the hydration, place slides in distilled Water for 00:05:00
5m
Sample Preparation after sectioning - Frozen
Sample Preparation after sectioning - Frozen
If frozen samples:
Remove from storage and let dry.
If embedded in OCT, put in distilled water for 00:05:00 at Room temperature .
If embedded in gelatin, put in PBS for 00:05:00 at 37 °C .
10m
Antigen Retrieval
Antigen Retrieval
Perform antigen retrieval using PT Module by Thermo Scientific with a cycle of pre-heat to 65ºC and heat to 95ºC for 20 minutes.
Note
This equipment, when optimized, allows you to skip the step of deparaffinization in this section, by putting the slides directly through the antigen retrieval step.
This equipment allows two buffers with different pH, in which we use EnVision FLEX Target Retrieval Solution High pHDakoCatalog #K8004 and EnVision FLEX Target Retrieval Solution Low pHDakoCatalog #K8005 , depending on the optimization of the antibody.
Blocking Endogenous Peroxidase and Total Proteins
Blocking Endogenous Peroxidase and Total Proteins
35m
Wash in 1X EnvisionTM Flex Wash Buffer Bio-opticaCatalog #Ref. K8007 3 times for 00:05:00 each
5m
Put slides in 200 mL of 100% EMSURE MethanolBio-opticaCatalog #1.06009.2500
Note
Extemporaneous solution. 100% methanol can be used up to 3 times.
Put 6 mL of Hydrogen Peroxide SolutionBio-opticaCatalog #7722-84-1 at 4 °C in the 100% EMSURE MethanolBio-opticaCatalog #1.06009.2500 with the slides
Cover the slide dish and incubate slides in the solution for 00:30:00
Note
Hydrogen Peroxide must be added each time when immersing the slides, to obtain a concentration of 3%.
30m
Wash in 1X EnvisionTM Flex Wash Buffer Bio-opticaCatalog #Ref. K8007 3 times for 00:05:00 each
Draw a limit on the edge of the tissue with the hydrophobic PAP Pen
Put Protein Block Serum free Bio-opticaCatalog #Ref. X0909 on the tissue
Incubation and Washing
Incubation and Washing
1h 54m 10s
Incubate with Primary antibody in the optimized concentration (if needed, dilute in Antibody DiluentBio-opticaCatalog #Ref. 8006 ) for 01:00:00 at Room temperature
Note
Normally, around 100μL of diluted antibody per section, depending on the size of the tissue.
1h
Wash in 1X EnvisionTM Flex Wash Buffer Bio-opticaCatalog #Ref. K8007 3 times for 00:05:00 each
Incubate with secondary antibody EnVisionTM Flex conjugated w/ HRP Bio-opticaCatalog #Ref. K4061 for 00:30:00 Room temperature
Note
Be sure to match the secondary antibody to the host of the primary antibody (ex: if using mouse anti-CD3 primary, use anti-mouse secondary)
30m
Wash in 1X EnvisionTM Flex Wash Buffer Bio-opticaCatalog #Ref. K8007 3 times for 00:05:00 each
Incubate with EnvisionTM Dako DAB+Bio-opticaCatalog #Ref. K3468 for 00:02:00 to 00:05:00
Safety information
After incubation, pour DAB excess on a paper towel and dispose on type IV waste, with tips and eppendorfs used, avoiding spills at any cost.
Clean the incubation chamber and surfaces with Bleach.
7m
Wash in distilled water for 00:05:00
5m
Counterstain and mount
Counterstain and mount
17m 10s
Counterstain with Harris hematoxylin for histologyBio-opticaCatalog #05-06004E for 00:00:10
Note
filter Hematoxylin before use
10s
Put slides in lukewarm running water for 00:05:00
5m
Dehydrate with 96% ethanol for 00:02:00
2m
Place in 100% ethanol for 00:02:00
2m
Clear in Xylene for 00:10:00
10m
Mount with appropriate mounting medium (i.e. Entellan ®Bio-opticaCatalog #1.00869 )
Note
Let the slides dry in a fume hood until complete polymerization of the mounting medium is achieved
Protocol references
1 - Magaki S, Hojat SA, Wei B, So A, Yong WH. An Introduction to the Performance of Immunohistochemistry. Methods Mol Biol. 2019;1897:289-298. doi: 10.1007/978-1-4939-8935-5_25. PMID: 30539453; PMCID: PMC6749998.
2 - Kim SW, Roh J, Park CS. Immunohistochemistry for Pathologists: Protocols, Pitfalls, and Tips. J Pathol Transl Med. 2016 Nov;50(6):411-418. doi: 10.4132/jptm.2016.08.08. Epub 2016 Oct 13. PMID: 27809448; PMCID: PMC5122731.