Question about Immunohistochemistry

[Fortyfive]

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hello,

Why do tissue sections stained with an antibody show up dark brown/red? I don't understand why they would be colored. Shouldn't they be clear because antibodies are colorless?

For ex:
http://static.enzolifesciences.com/fileadmin/files/image/ADI-950-112_IHC.gif
http://library.med.utah.edu/WebPath/jpeg2/AIDS011.jpg
https://upload.wikimedia.org/wikipe...human-Xenograft-Immunohistochemistry_HER2.png

I understand the basis behind immunofluorescence and I know why you can get colorful images. If someone could help me understand how the colors in the previous images are produced, I would really appreciate it! Thanks!

 

[SurfingDoctor]

The principles are the same.

In immunofluorescence the secondary antibody (that binds to the primary antibody that binds to the protein/target of interest) has a fluorescent tag (FITC, Cy5, Texas Red, etc.) that you can see under a specific wavelength of light.

In immunohistochemistry, the secondary antibody is conjugated to a enzyme (eg. avidin-horseradish peroxidase) that reacts with an substrate (eg. 3,3'-diaminobenzidine) that produces color that is visible to the naked eye.

 

[Fortyfive]

Ohh, thank you so much! I never learned about conjugated substrate/enzyme reactions. I will read more about it. My histology professor never explained why there were colors. I appreciate your explanation! Thank you again!