1.5 Immunofluorescence
Reagents and Equipment
 Labelled or un-labelled primary and secondary antibody (keep at 4oC)
 Fix = PBS + 2% Formaldehyde (from stock 38%) – Make up fresh on day of use.
 Wash = PBS + 5% FCS
 Saponin 10% in PBS (detergent, makes cells permeable)
 Cover slip with adherent cells or non-adherent cells adhered to poly-l-lysine
coating.
 Fluoromount G
 Nail varnish
 Parafilm
 Petri dish
 Tweezers
 Twelve well dishes
Procedure
1. Plate cells on cover slips in 6 or 12 well dish. Transfect or treat as required.
2. Make up sufficient amounts of fix (PBS + 2% Formaldehyde) enough for 1.5ml
per slip. For 10ml:
 Volume required = (End concentration/Start concentration) x End volume
= 2 %/38% x 10ml
= 0.526ml
= 526µl
 9.5ml PBS + 526µl Formaldehyde.
3. Make up sufficient amount of wash (PBS + 5% FCS) for all the washes required.
For 40ml:
 Volume required = 5%/100% x 40ml
= 2ml.
 38ml PBS + 2ml FCS.
4. Aliquot 1.5ml of fix for each cover slip into individual wells in a 12 well plate.
Label the plate appropriately.
5. Transfer each cover slip into the wells containing the fix. Using tweezers keep the
slip the same side up and touch the slips onto a piece of tissue to remove the
excess media. Incubate at room temperature for 10 minutes.
6. Aspirate the fix and add 1ml wash to each well, incubate at room temperature for
5 minutes. Repeat a further 2 times.
7. Prepare 1o Antibody Mix (total volume 50µl for two slips. 15µl required for one
slip, but 50 µl is the minimum volume, for more than one slip make up 15µl for
each slip + 20µl excess e.g. for 10 slips: 170µl). NB Each antibody has different
dilution factors. For example, GM130 has a dilution factor of 1:50 (for 50l):
 Ab
1µl
 Wash
48µl
 Saponin 1µl (stock solution 10%, end concentration 0.2%)
Tap tube, then pulse centrifuge (to collect antibody mix).
8. Pipette 15-25µl of 1oAb mix onto a piece of parafilm in a Petri dish, it is easier to
arrange the drops in the same maner as they are plated in the 12-well plate to
avoid confusion. Add a wet piece of tissue in the corner of the dish to maintain
humidity and prevent drying out.
9. Using tweezers remove the slip from the well and touch onto a piece of tissue to
remove the excess wash. Invert the slip onto the Ab mix drop in the dish.
10. Incubate for 1 hour at room temperature.
11. Remove the slip in the same way as before and place in well (containing 1ml
wash) cell side up (i.e. side that was exposed to antibody). It is easiest to use a
yellow pipette tip to help pick up the coverslip. Leave for 5 minutes.
12. Aspirate wash and repeat wash step 2 further times.
13. Prepare 2o Ab Mix: (for 50µl)
 Ratio
Rh α Mouse dilution factor 1:250.
 Ab
0.25µl
 Wash
49µl
 Saponin 1µl
Tap tube and pulse centrifuge for 10 seconds to collect the antibody mix.
14. In the same way as the 1oAb mix, pipette 15-25µl of 2oAb mix onto a piece of
parafilm in a Petri dish with a wet tissue in the corner. Remove the cover slip and
invert onto Ab mix drop.
15. Incubate for 1 hour, at room temperature, in the dark (i.e. wrap in foil).
16. Repeat 11 and 12.
17. Aspirate wash and leave slips in 1ml PBS for 5 minutes (can be left for longer).
18. Put 25µl Fluoromount G onto microscope slide, being careful to avoid bubbles-to
avoid this apply a little pressure to the pipette before you remove it from the
liquid. Make sure the slides are labelled before you start. If using non-adherent
cells then add DAPI to the Fluoromount G (add at approximately 1:100).
19. Remove the slip from the well as before and invert slip onto fluoromount G. Turn
the whole slide over onto a tissue to remove excess fluoromount G and then use
nail varnish to seal the edges of the slip.
20. After allowing the nail varnish to dry, store in the fridge, in the dark (wrap in foil
or put in box) before imaging.