Why Immunohistochemistry requires a conjugated enzyme, whereas immunofluorescence does not. Explain the difference?
Dear Shrikant,
actually, strictly speaking, immunofluorescence is a type of immunohistochemistry.
In case if you use enzyme-linked Ab, you get signal as a result of enzymatic reaction - a substrate molecule is being degraded by the enzyme, and a colored molecule is produced.
In case if you use fluorescent-labeled Ab, you then irradiate your samples and get signal as fluorescence.
I think this picture should help understand these processes better.
Dear Shrikant,
actually, strictly speaking, immunofluorescence is a type of immunohistochemistry.
In case if you use enzyme-linked Ab, you get signal as a result of enzymatic reaction - a substrate molecule is being degraded by the enzyme, and a colored molecule is produced.
In case if you use fluorescent-labeled Ab, you then irradiate your samples and get signal as fluorescence.
I think this picture should help understand these processes better.
The detection methods differ between those two staining methods.
In IHC you usually detect your staining results with brightfield microscope. Therefor you want a chromophore that is visible at the wavelength(s) of visible light.
This is usually being achieved by letting a horseradish peroxidase/ or alkaline phosphatase-conjugated enzyme bind to your antibody (for the protein of interest) followed by adding a chromophore which changes the color after the reaction with the conjugated enzyme.
In IF you have a fluorophore coupled to your antibody. A fluorophore gets excited via a certain wavelength and emits light with higher wavelength. With the help of the filtersystem within a Fluorescence microscope you can filter the incoming wavelenghts so you can see (almost only) the structure to which your antibody has bound.
Hi
I agree with researchers here , See link here for more info
Good Luck
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2362713/
https://bio.libretexts.org/TextMaps/Map%3A_Microbiology_(OpenStax)/20%3A_Laboratory_Analysis_of_the_Immune_Response/20.4%3A_Enzyme_Immunoassays_(EIA)_and_Enzyme-Linked_Immunosorbent_Assays_(ELISA)
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