Immunofluorescence

Immunofluorescent Staining Protocol
Plating
1. Clean desired number of coverslips (Corning 22x22mm Square #1.5) with 100%
ethanol
2. Place individual coverslips in the wells of a 6-­‐well plate
3. Add cells and incubate overnight
Fixation
1. Wash cells with 1X PBS quickly to remove medium
2. Fix cells using 3.7% paraformaldehyde (make fresh each time)
3. Incubate in paraformaldehyde for 10 minutes at room temperature
4. Wash cells with 3 quick rinses of 1X PBS to remove paraformaldehyde
Permeabilization
1. Incubate cells in PBS + 0.2% Triton X-­‐100 for 2 minutes at room temperature
(punches holes in the membrane to allow antibodies to permeate the cell)
2. Wash cells with 3 quick rinses of 1X PBS to remove detergent
Blocking
1. Incubate permeabilized cells with 5% goat serum for 30 minutes at room
temperature (helps to prevent non-­‐specific intracellular antibody binding –
background staining)
Staining
*If possible, mix primary antibodies into 5% goat serum, this will also help to cut down
on background staining
*Concentrations for antibodies will vary, see manufacturers suggestions or previous lab
protocols to determine which concentration works best
1. Incubate cells in 1X PBS* + primary antibody for 1hr at room temperature
2. Wash cells 4 times with PBS at room temperature (10 minutes each wash)
3. Incubate cells in 1X PBS + secondary antibody for 1hr at room temperature IN THE
DARK to prevent photo-­‐bleaching from occurring (when using fluorescent
molecules, be sure to limit the time spent in direct light to a minimum)
4. Wash cells 4 times with PBS at room temperature (10 minutes each wash) – if
staining with DAPI add to the first wash (have DAPI at 11mM, dilute to 300uM – use
at 1:1000 dilution)
Mounting
1. Place a drop of mounting medium onto the slide and carefully overlay glass
coverslip, CELL SIDE DOWN, onto the medium (start by making contact between
the medium and coverslip at one end of the coverslip and gently lay the coverslip
down onto the medium – this helps to keep the bubble formation to a minimum)
2. Allow slides to dry overnight (2-­‐3 hours at a minimum) IN THE DARK at room
temperature
3. Visualize by fluorescence microscopy

3.7% Paraformaldehyde
dilute 37% formaldehyde 1:10 into PBS (needs to be made fresh each time)
PBS + 0.2% Triton X-­‐100
2X PHEM Buffer (500mL)
18.14g PIPES
6.5g HEPES
3.8g EGTA
0.99g MgSO4
pH to 7.0 with 10M KOH
filter and store at 4°C
20% Goat Serum (Heat-­‐Inactivated)
1. Heat desired volume of goat serum needed to make serum solution at 60-­‐70°C
for 1hr
2. Allow serum to cool
3. Add appropriate volume of 2X PHEM buffer and sodium azide to 0.05%
4. Filter through 0.22uM filter and store at 4°C
5. Dilute for blocking