EZQuant–ELN Knowledge Base

General immunohistochemistry protocol	Table of Contents

General Note:

Protocols for immunohistochemistry vary widely, according to different antigens and their recognition by their antibodies. Some epitopes can be harmed by high temperatures and organic solvents, while others cannot survive freeze and thaw. For successful immunohistochemistry it is crucial to try a number of different techniques and compare the results. If possible, obtain the procedure details used by other researchers for a specific antibody.

 
The presented protocol is suitable for mounted frozen sections:

• warm slides to room temperature for 30 minutes.
• Wash 5 min in PBS.

When horseradish peroxidase (HRP) is used, quenching of endogenous peroxidases is usually beneficial:

• Incubate slides in 3% hydrogen peroxide in methanol or PBS for 30 min at room temperature.
• Wash 2 x 5 min in ddH₂O.

• Wash 5 min in PBS.

Tissue permeabilization (optional):

• Incubate slides in PBS+0.1% Tween 20 (PBS-T) for 5 min.
• Wash 5 min in PBS.

Blocking:

• Draw boundaries of the sections using a PAP pen and let it dry for a few seconds.
• Block sections with 10% serum of the species in which the secondary antibody was prepared. Incubate in a humidified chamber for 1 hr at room temperature.
• Discard blocking solution by shaking it off the slide (do not wash the slide).

Primary antibody:

• Dilute the antibody in PBS+2% BSA or 2% serum. The exact dilution should be determined empirically.  
• Use Kimwipes to remove excess liquids from the slides, then add the antibody directly onto the sections.
• Incubate overnight in a humidified chamber at 4^oC.
• Wash 3 x 5 min with PBS (or PBS-T).

Secondary antibody:

• Incubate sections with the appropriate biotinylated secondary antibody for 1 hr at room temperature.
• Wash 3 x 5 min with PBS (or PBS-T).

Streptavidin-HRP:

• Prepare streptavidin-HRP solution as recommended by the manufacturer.  
• Incubate sections in streptavidin-HRP for 30 min at room temperature.
• Wash 3 x 5 min with PBS (or PBS-T).

DAB:

• Incubate sections in fresh DAB solution (10mg DAB + 20µl 38% H₂O₂ in 20ml 0.1M Tris, pH 7.2). Stop the reaction by immersion in ddH₂O.
•  Rinse slides in running tap water for 5 minutes.

Dehydrate and cover:

·         Dehydrate slides:
1 min ethanol 70%
1 min ethanol 95%
1 min ethanol 100%
2 x 5 min Xylene (in a chemical hood).

·         Apply 2 drops of mounting medium on the slide and cover with a coverslip.