4. INTRODUCTION
Immunofluorescence is a technique
allowing the visualization of a specific
protein or antigen in tissue sections by
binding a specific antibody chemically
conjugated with a fluorescent dye.
5.
6. TYPES
Direct immunofluorescence: staining
in which the primary antibody is labeled
with fluorescence dye,
Indirect immunofluorescence: staining in
which a secondary antibody labeled
with fluorochrome is used to recognize
a primary antibody.
7.
8. A different primary antibody is used for each
target protein. Variable part of primary
antibody binds to specific part of target
protein.
The secondary antibody binds to the constant
part of the primary antibody. Therefore a
sample of the same batch of secondary
antibody can bind to many different primary
antibodies.
10. Repeat washing
Primary antibody solution against target
protein molecule
Secondary antibody, add with florescent
marker
11.
12.
13. Detection Procedure
Primary antibody against target protein
Than secondary antibody
MOUNTING SOLUTION
o It could be with DAPI or without DAPI.
DAPI SOLUTION
It is a solution that stains the chromatin material of
nucleus.
DAPI, (4',6-diamidino-2-phenylindole) is a
fluorescent stain that binds strongly to A-T rich
regions in DNA, to label DNA.
14. DAPI is a blue florescent dye, It emits blue
colour florescence.
15. Applications
Immunofluorescence can be used on tissue
sections, cultured cell lines, or individual cells,
and may be used to analyse the distribution
of proteins and small biological and non-
biological molecules.
It also play a key role in the diagnosis of
autoimmune disorder.
16. Limitations
Quality and concentration of the antibody.
Proper handling of the specimen.
Choice of secondary antibodies.
Fluorophores undergoes photobleaching as
they are exposed to light.
