Immunohistochemistry protocol optimized at iMM-JLA
Joana G Antunes, Ana M Biscaia Santos, Ana M Cristóvão Pinto, Ana R Pires
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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).
Before start
If FFPE - begin protocol after adhering sections to the slides for 1h at 60ºC
Steps
Preparation of Wash Buffer
Measure 20mL of 20X
Make up with distilled water to 1L of total volume
Sample Preparation after sectioning - FFPE
Deparaffinize slides with Xylene for 0h 10m 0s
Change Xylene and deparaffinize for another 0h 10m 0s
Place slides in Ethanol 100% for 0h 5m 0s
Place slides in Ethanol 96% for 0h 5m 0s
Place slides in Ethanol 70% for 0h 5m 0s
To complete the hydration, place slides in distilled Water for 0h 5m 0s
Sample Preparation after sectioning - Frozen
If frozen samples:
Remove from storage and let dry.
If embedded in OCT, put in distilled water for 0h 5m 0s at Room temperature .
If embedded in gelatin, put in PBS for 0h 5m 0s at 37°C.
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.
Blocking Endogenous Peroxidase and Total Proteins
Wash in 1X 0h 5m 0s each
Put slides in 200mL of 100%
Put 6mL of 4°C in the 100%
Cover the slide dish and incubate slides in the solution for 0h 30m 0s
Wash in 1X 0h 5m 0s each
Draw a limit on the edge of the tissue with the hydrophobic PAP Pen
Put
Incubation and Washing
Incubate with Primary antibody in the optimized concentration (if needed, dilute in 1h 0m 0s at Room temperature
Wash in 1X 0h 5m 0s each
Incubate with secondary antibody 0h 30m 0s Room temperature
Wash in 1X 0h 5m 0s each
Incubate with 0h 2m 0s to 0h 5m 0s
Wash in distilled water for 0h 5m 0s
Counterstain and mount
Counterstain with 0h 0m 10s
Put slides in lukewarm running water for 0h 5m 0s
Dehydrate with 96% ethanol for 0h 2m 0s
Place in 100% ethanol for 0h 2m 0s
Clear in Xylene for 0h 10m 0s
Mount with appropriate mounting medium (i.e.
