DBL 04/2007
Supplemental Information for Immunocytochemistry Protocol
Day One:
1.
Fix cells in 4% formalin for 10 minutes. Dilute 1:10 in PBS. If more needs to be made, dilute
1:10 in PBS (e.g. if making 50mL 10% formalin, add 2mL 37% formaldehyde to 48mL PBS).
2.
Aspirate off formalin and wash once in 0.1% Triton X-100 for 5 minutes. Use stock Triton X-100
and dilute 1:1000 in PBS (e.g. if making 50mL 0.1% Triton X-100, add 50µL stock Triton to
49.95mL PBS).
3.
Aspirate off Triton X-100 and wash 3 times in 1X PBS for 5 minutes each. During these washes,
prepare 1% and 8% BSA solution, as they take a while to dissolve.
4.
Block in 8% BSA for 25 minutes. To calculate the percentage, follow this rule:
1% =
1g solid
100mL solution
Thus for 50mL 8% BSA, add 4.0g BSA to 50mL PBS. For 50mL 1% BSA, add 0.5g BSA to 50mL
PBS. Stir slowly on magnetic stir plate—don’t want protein to degrade by overaggressive
agitation.
5.
Make primary antibody solution in 1% BSA. Check recommended dilutions in the notebook
labeled “Antibodies.” If using a mixed culture, co-stain using anti-Map2 (1:1000 dilution). This
allows for distinction between neurons and glial cells. The total volume of primary antibody
solution depends on the number of coverslips. In your notebook, note the source of each
antibody, the catalog #, and the lot #.
Double Staining: Be sure the antibody solution is based on the concentrations of each antibody
(i.e. HSF1 is 1:10,000 while HSP70 is 1:1000) and that the total volume applied to each
coverslip creates the proper concentration. Record dilutions in your notebook.
6.
Using forceps, gently move the coverslips to pedestals. The pedestals should be placed in a
tray. Add a small amount of water to the pedestal containers to decrease evaporative loss;
also, line the edges of the tray with damp paper towels. Make sure that the coverslips remain
cell-side up. Record which coverslips are on which pedestals.
7.
Add 50µL primary antibody solution to each coverslip. Cover the tray and incubate at 4ºC
overnight. Save the 24 well plate that the cell came from until cells are mounted.
Day Two:
8.
Bring the coverslips back to room temperature for 5 minutes.
9.
Place the coverslips back in their respective wells, and wash 6 times in 1 mL of PBS for 5
minutes each. During these washes, prepare secondary antibody solution.
10.
Make secondary antibody solution in 1% BSA. These antibodies can be found in the lab’s -20ºC
freezer. When choosing a concentration, recall that the secondary antibodies are stored at a
1:1 dilution with glycerol. In your notebook, note which fluorescent tag is on which antibody.
Be sure the antibodies match the animal used for the primary antibody staining. Make up 300
µL of secondary ab per well to ensure sufficient quantities.
11.
Add 300µL of the antibody solution to each well. Incubate at room temperature for 1 hour.
Place the plate in the dark (e.g. in a drawer or wrapped in foil).
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DBL 04/2007
12.
Following the incubation, wash the wells 5 times with PBS for 5 minutes each.
13.
Remove the PBS from the last wash, and stain the cells using DAPI at working concentration of
1µg per mL of PBS. Concentrated DAPI and working stocks can be found in the lab refrigerator
in labeled Eppendorf tubes. Dilute 1:1000 in PBS. Add 50µL to each coverslip. Place in the dark
for 10 minutes.
14.
Remove the stain and wash 3 times in PBS for 5 minutes each. During each wash, place the
well-plate in the dark.
15.
Prepare microscope slides to mount the coverslips, using 3 coverslips/slide at most. Use a
different slide for each different cell group or experimental condition. Label in pencil the
following information: the names of the cell groups (this should be written on the well-plate),
primary antibodies used, secondary antibodies used, the date, and your initials. Place 1 drop of
n-propyl gallate (stored in the -4ºC refridgerator in an Eppendorf tube) on the slide to mount 1
coverslip. Do not use a lot of n-propyl gallate.
16.
Mount the coverslips onto microscope slides cell side down. Use forceps to gently pull up the
coverslips from their edges; a needle may be helpful in lifting up the coverslips. Aspirate off
any excess n-propyl gallate.
17.
Let the slides dry in the dark, storing them in a cardboard microscope slide book.
18.
Seal coverslips with clear finger nail polish, applying it only around edges of each coverslip.
Allow polish to dry.
19.
Store at -20˚C in the cardboard book until pictures can be taken. You can not take pictures
the same day on which the slides are sealed.
Notes:
 Be sure you write all information about the primary antibody (lot #, catalog #, species).
 In double staining, ensure that you match the fluorescent tag with the appropriate species (i.e.
do not use two mouse antibodies in the same staining)
Total time:
Day One - ~1 hour; Day Two- 3-4 hours.
Purpose:
To visualize protein expression in primary cultures
Equipment:
1 mL pipette, 20 µL pipette, 100 µL pipette, 24 well plate, glass pipettes, Formalin
solution, BSA solution, primary antibody, secondary antibody, DAPI, forceps, needle, n-propyl gallate,
clear fingernail polish.
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