Immunofluorescence protocol
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Immunofluorescence protocol
Solutions and reagents
All the solutions should be prepared by using either a reverse osmosis deionised water or water of similar grade.
All the incubations should be performed at 20-25°C unless stated otherwise.
Phosphate buffered saline (PBS)
Formaldehyde 4%, Methanol free
Blocking buffer
Antibody dilution buffer
Fluorophore conjugated secondary antibody
Fixation for fixed frozen tissues
1. Block the specimen in blocking buffer for 1 hr.
2. During the blocking, prepare the primary antibody in the dillution buffer.
3. Aspirate the blocking solution, then add the diluted primary antibody.
4. Overnight incubation at 4°C.
5. Three five minute rinses using PBS.
6. Incubate the specimen in the fluorochrome conjugated secondary antibody for 1-2 hrs. (Note: make sure it is protected from light to avoid photobleaching)
7. Three five minute rinses using PBS.
8. Counterstain.
9. Mount the sample for imaging.
10. If the samples will be stored for long periods of time, keep them away from light and at 4°C.
Fixation for cultured cell lines or unfixed frozen tissues
1. Cover the specimen in Formaldehyde 4%.
2. Let the specimen fix for 15 min at room temperature.
3. Three five minute rinses using PBS.
4. Proceed with the Immunostaining steps outlined in the previous section.