UPDATED 29. 11. 2023 15:40 by Hana Hajšmanová Introduction This technique uses the specificity ofantibodiesto theirantigento targetfluorescentdyesto specificbiomoleculetargets within a cell, and therefore allows visualization of the distribution of the target molecule through the sample. Immunofluorescence is a widely used example ofimmunostaining(using antibodies to stain proteins) and is a specific example ofimmunohistochemistry(the use of the antibody-antigen relationship in tissues). This technique primarily makes use of fluorophores to visualize the location of the antibodies. Materials
- If coating with 0,1% gelatine, add500µLper well and leave it for 30 minutes, then suck up the gelatine (erratum Janek - on coverslip, 10 min, aspirate let dry)
- If coating with 0,01% poly-L-lysine, incubate the coverslips for 5 minutes, remove the poly-L-lysine and leave to dry 2. Seed the cells. Density approx.60,000 per well of 24-well plate 3. Transfect or treat the cells 24 hours after seeding Staining 4. Insert a clean 13mm coverslip before seeding cells In one well of the 24 well plates. (https://microscopy.duke.edu/guides/glass-coverslip-cleaning) 5. Grow cells in 24 well plate (insert 13mm cover slip before seeding cells) 6. Remove medium, wash cell withPBS1X 7. Fix in freshly diluted 4% formaldehyde (cc.200µL/well) inPBSfor 15 min @RT 8. Wash 3X PBST (after this step you can store the cells in the fridge overnight) 9. Block 45min in PBTA @RT (200µL/well) 10. Incubate O/N with primary antibodies in PBTA @ 4 C (200µL/well; dilutions may vary) Primary antibodies can be reuse. 11. Wash in 3 changes of PBST, 5-10 min each. 12. Block for 30 min with PBTA @RT (Not Necessary) 13. Incubate 1 hour with secondary antibodies in PBT - withoutNaN3@RT. Protect from the light (cc.200µL/well). 14. Wash in 3 changes of PBST, 10 min each. Include DAPI/Hoecht (1µg/mL) in the last wash.(possible to reuse DAPI) 15. Replace PBST withPBS; wash 3X 10 min withPBS 16. ReplacePBSwith mounting solution (Mowiol) or DAPI Mounting media. (17-20µLof Mowiol)