Immunofluorescence / IF / ICC Protocol

According to the steps of experiment, immunofluorescence includes preparation of cells or tissues, fixed cells or tissues, permeabilized, closed and incubated with anti incubation second antibody, mounting and imaging steps.
Comparison between direct and indirect methods:Direct method is less sensitive, but the sensitivity is not high, and the indirect method, because of the surface of the antibody immunoglobulin molecules, can be combined with a number of fluorescent antibody molecules, and play a role in amplification, the sensitivity is higher than the direct method 5~10 times. There are many factors that are involved in the process of non - specific fluorescence, which is more than the direct method.
Every step of the immune fluorescence is critical, and every step error is either false or unreliable. So in general, laboratories or companies will be strictly regulate the operation of the immune fluorescence steps and processes, every step to ensure that no error, to get a good result .

Immunofluorescence / IF / ICC flow chart

Immunofluorescence / IF / ICC Antibody
Immunofluorescence / IF/ICC Antibody Features
Immunofluorescence / IF/ICC Products Center
Immunofluorescence / IF / ICC Technology Center+
- Immunofluorescence / IF / ICC Introduction
What is Immunofluorescence (IF)?
What is Immunocytochemistry (ICC)?
Difference between Immunocytochemistry / ICC and Immunofluorescence / IF
Principle of Immunofluorescence
Advantages of Immunofluorescence / IF/ICC
- Immunofluorescence / IF / ICC Protocol
Immunofluorescence protocol (IF protocol)
Immunocytochemistry protocol (ICC protocol)
Immunofluorescence / IF/ICC-Immunostain protocol
- Immunofluorescence / IF / ICC FAQ
Does the immunefluorescence antibodies be used to other application,WB,IP,IHC,FC or CHIP?
What is the specimen commonly used in immunofluorescence l staining experiments?
What is the difference Immunocytochemistry / ICC and Immunofluorescence / IF?
Why the immune fluorescence result has non-specific staining?
How to select the immune fluorescent antibody?
indirect IF or dierect IF?
What is a good result for membrane protein?
What is a good result for cytoplasm protein?
What is a good result for nuclear protein?
How to choose second antibody for IF?
Which is the best method, IF or IHC ?
- Immunofluorescence / IF / ICC Troubleshooting
Positive Control is negative or very weak
Negative Control is positive
Why the immune fluorescence result is no staining?
Why the immune fluorescence result has high background?
Why the immune fluorescence result has non-specific staining?
High background staining showed false positive or negative results
How to choose second antibody for IF?
How to preserve the immuno fluorescence antibody?
Why the antibody without IF application can't used for IF test?
Reactions are stronger on the edges of the well in comparison to the center
- Immunofluorescence / IF / ICC Tips
Antibodies determine success or failure of IFF/ICC
Cell fixation and permeabilization
Cell Use an appropriate cell density
Cell Multiple staining
Cell Reducing background
-Examples of good or bad IF/ICC Results