Question

What are some strengths, weaknesses, and techniques of immuno-fluorescence microscoy?

Answer 1

Immunofluorescence (IF) microscopy is a widely used example of immunostaining and is a form of immunohistochemistry based on the use of fluorophores to visualize the location of bound antibodies. It is a particularly robust and broadly applicable method generally used by researchers to assess both the localization and endogenous expression levels of proteins of interest.

Limitations: fixing cells, molecules on the outside of the cell membrane or in the supernatant can be bound by the antibodies which allow for living cells to be stained, and molecules in cell membrane must be stained after fixation which may make protein cross-linked and false negative or false positive signals due to non-specific binding.

Advantages:

1. Clear positioning. This assay can give research the clear subcellular localization of molecules.
The expression of molecules can be observed directly.
2. High specificity. The preparation of sample can effectively protect the natural structure of the antigen
3. High sensitivity. The target with low expression can be detected with immunofluorescence / IF/ ICC.
4. Easy to operate. This assay can be used vivo experiments.
5. Multiple staining. The multiple targets can be detected in the same sample.

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