Immunofluorescence protocol

Keep samples (usually a coverslip with adherent cells) submerged in liquid at all times until sample preparation is completed and has been attached to a slide with Gelvatol.  Store completed samples at 4oC in the dark when not in use. Stained samples may be rescoped for several months if stored this way (depending on the antibodies and fluorophores used).

Controls: All experiments should include a sample that has been treated to control for non-specific primary and secondary binding. Some controls include:

*Incubation with secondary antibody only (to determine non-specific staining by the secondary antibody).

*Non-immune serum or isotype Ig (matching the primary antibody).

*Infrequently, some cells display autofluorescence, you may need to have a non-processed sample to control for this.

1.    Remove medium, follow with 3 washes of 1xPBS

2.    Fix sample in 2% paraformaldehyde made in PBS for 15 minutes at room temperature.  Wash monolayer 2 times in PBS to remove residual paraformaldehyde, store samples submerged in PBS at 4oC until use.  If sample(s) already have a fluorescent label (e.g. Mitotracker, Lysotracker, Di-I-AcLDL, etc…), then minimize exposure to light. Make sure these labels are compatible with this particular fixation and permeabilization protocol.

3.    Permeabilize cells with 0.1% Triton X-100 made in PBS solution for 15 min. Different detergent permeabilization protocols may be necessary depending on your system.  Please contact a member of CBI with details of your system.

4.    Wash monolayer 3 times with PBS.

5.    Wash monolayer 5 times with a PBS+ 0.5% BSA (PBB).

6.    BLOCK with 2% BSA for 45 minutes. Alternative blocks are 20% serum in PBB from the species in which your secondary antibodies are made (e.g. if you secondary antibody is made in goat, use 20% goat serum. Likewise if your secondary is made in donkey, use 20% donkey serum).

7.    Wash monolayer 5 times with PBB.

8.    Primary antibody: Dilute to the desired concentration in PBB (vortex gently, spin down for 5 min at 10-12K RPM to get rid of aggregates). Gently drop 70-100 µl antibody solution over the section (so the section is all covered). Incubate for 60 minutes.. Longer primary antibody incubations may be necessary and must be determined empirically in your system.  Initial antibody optimization should be accomplished in a range up to 5ug/ml antibody in PBB.  If longer incubation times are necessary, place a piece of wet paper towel in your slide box (or chamber) with your samples to minimize evaporation of your primary antibody solution.  In some cases overnight incubations at 4oC are necessary, you must put your samples in a humid environment (wet paper towel within slide box), avoid disturbing the sample volume on your section. Primary antibodies can be added together but the host must be from 2 different species.

9.    Wash monolayer 5 times with PBB.

10.    Secondary antibody for 60 minutes (made in PBB, vortex, spin down at 10-12K RPM for 5 minutes, avoid using solution from the bottom of the vial).  The secondary antibody step should be kept to within an hour.  You may add fluor-conjugated phalloidin (for F-actin counterstain) or Draq5 (a far-red nuclear stain from Biostatus) to your secondary solution. Secondaries can be added together if using more than one, but they must be against two separate species.

11.    Wash monolayer 5 times with PBB

12.    Hoescht stain for 30 seconds  Hoechst (Sigma, cat no. B-2883) is made at concentration of 1mg/100ml in dH2O and is stored in the dark at 4oC when not in use. Caution! This substance is carcinogenic and toxic to skin!!

13.     Wash monolayer 3 times with PBS.

14.    Adhere coverslip with gelvatol to a slide (avoid bubbles) and store samples horizontally at 4oC with minimal exposure to light until use.  If using coverslip-bottomed chamber slides or Met-Tek dishes and an inverted scope, gelvatol is not necessary. Store cells in PBS at 4oC and do not allow them to dry out at any time until scoped.
Gelvatol is water soluble, if needed coverslip may be gently removed by submerging sample in PBS and allowing coverslip to lift without resistance from the slide. Recipe for Gelvatol on protocol website.

General recipes:
BLOCK (PBS+2% BSA) = 2g BSA per 100mL 1xPBS
PBB (PBS+0.5% BSA)= 0.5g BSA per 100ml PBS

*Other fixation protocols:
In some cases antibodies won’t work on paraformaldehyde fixed epitopes.  Other fixations include 100% cold methanol or acetone (solvent stored at -20oC). Do not use acetone if your coverslips are polystyrene or stored in polystyrene tissue culture dishes. Acetone dissolves this plastic.  Move coverslips to another type of container if this type of fixation is necessary.

1. Immerse coverslips in cold solvent for 10 min.

2. Remove and let air dry.

3. Store these at 4oC or -20oC until processing.

4. Rehydrate cells with PBS and wash 3 times.

5. Proceed as above for paraformaldehyde fixed cells.
Note: Cells fixed in this manner will NOT counterstain with phalloidin.

There are many other permutations and other options available when it comes to immunofluorescence protocols. The ones described above are basic and work for most applications. However, if you have any questions, or are experiencing problems, please contact a CBI staff member for assistance. Also, may websites discuss antigen retrieval in fixed tissues, such as: http://www.ihcworld.com/_protocols/epitope_retrieval/en_bloc.htm.