TITLE: Immunohistochemistry protocol optimized at iMM-JLA
AUTHORS: Ana M Biscaia Santos, Ana M Cristóvão Pinto, Ana R Pires, Joana G Antunes

[DESCRIPTION]
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]
SECTION: Preparation of Wash Buffer
1. Measure 20 mL of 20X   

SECTION: Preparation of Wash Buffer
2. Make up with distilled water to 1 L of total volume

SECTION: Antigen Retrieval
10. Perform antigen retrieval using PT Module by Thermo Scientific with a cycle of pre-heat to 65ºC and heat to 95ºC for 20 minutes.

 

SECTION: Counterstain and mount
28. Clear in Xylene for 10 min  

SECTION: Counterstain and mount
26. Dehydrate with 96% ethanol for 2 min

SECTION: Counterstain and mount
25. Put slides in lukewarm running water for 5 min 

SECTION: Counterstain and mount
24. Counterstain with   for 10 s   
 


SECTION: Incubation and Washing
23. Wash in distilled water for 5 min

SECTION: Incubation and Washing
22. Incubate with    for 2 min to 5 min 

 

SECTION: Incubation and Washing
21. Wash in 1X   3 times for 5 min each

SECTION: Incubation and Washing
20. Incubate with secondary antibody   for 30 min  Room temperature 

 

SECTION: Incubation and Washing
19. Wash in 1X   3 times for 5 min each

SECTION: Incubation and Washing
18. Incubate with Primary antibody in the optimized concentration (if needed, dilute in  ) for 60 min at Room temperature 
 

SECTION: Blocking Endogenous Peroxidase and Total Proteins
17. Put   on the tissue

SECTION: Blocking Endogenous Peroxidase and Total Proteins
16. Draw a limit on the edge of the tissue with the hydrophobic PAP Pen 
SECTION: Blocking Endogenous Peroxidase and Total Proteins
14. Cover the slide dish and incubate slides in the solution for 30 min 

 

SECTION: Blocking Endogenous Peroxidase and Total Proteins
13. Put 6 mL of   at 4 °C in the 100%   with the slides

SECTION: Blocking Endogenous Peroxidase and Total Proteins
12. Put slides in 200 mL of 100%  

 

SECTION: Blocking Endogenous Peroxidase and Total Proteins
11. Wash in 1X   3 times for 5 min each

SECTION: Sample Preparation after sectioning - FFPE
3. 
Deparaffinize slides with Xylene for 10 min 
SECTION: Blocking Endogenous Peroxidase and Total Proteins
15. Wash in 1X   3 times for 5 min each

SECTION: Sample Preparation after sectioning - Frozen
9. If frozen samples: 
Remove from storage and let dry. 
If embedded in OCT, put in distilled water for 5 min at Room temperature .
If embedded in gelatin, put in PBS for 5 min at 37 °C.

SECTION: Sample Preparation after sectioning - FFPE
4. Change Xylene and deparaffinize for another 10 min 
SECTION: Sample Preparation after sectioning - FFPE
5. Place slides in Ethanol 100% for 5 min

SECTION: Sample Preparation after sectioning - FFPE
6. Place slides in Ethanol 96% for 5 min

SECTION: Sample Preparation after sectioning - FFPE
7. Place slides in Ethanol 70% for 5 min 

SECTION: Sample Preparation after sectioning - FFPE
8. To complete the hydration, place slides in distilled Water for 5 min

SECTION: Counterstain and mount
27. Place in 100% ethanol for 2 min

SECTION: Counterstain and mount
29. Mount with appropriate mounting medium (i.e.  )