Exercise 2.7 - Immunofluorescent Localization of Tubulin
- Coverslip cultures of an appropriate monolayer cell line
- Phosphate buffered saline (PBS)
- Acetone/Methanol (absolute) in a 50:50 volume mixture
- Rabbit anti-tubulin (or other primary antibody to tubulin)
- FITC-labeled goat anti-rabbit (or secondary antibody to match the primary)
- Fluorescence Mounting Media
- Fluorescent Microscope equipped with 490 nm excitation filter and 515 nmbarrier filter
- Kodachrome film or equivalent color slide film (Kodak Tri-X or Ilford HP400 may be substituted for black and white photography)
- Set up a coverslip culture of an appropriate cell line
24 hours prior to the lab. This is best accomplished by dry
sterilization of #1 coverslips which are subsequently placed
in plastic tissue culture plates. Cells are placed on the
coverslips with sufficient media to cover and allowed to grow
for 24 hours. There should be sufficient cells to view
comfortably, but they should not be crowded on the slide.
- Remove the coverslip from the culture plate and dip several
times in a beaker of phosphate buffered saline (PBS) to rinse
off the culture media. Drain, but do not allow to dry.
- Immediately immerse in a 50:50 mixture of acetone/methanol
at room temperature. Allow the coverslips to remain in the
acetone/methanol for 2 minutes.
- Remove the coverslips from the acetone mixture and rinse
2X with PBS.
- Prepare a 1/40 anti-tubulin dilution using PBS. PbS
alone may be used or better, augment the PBS with 3% (w/v)
Bovine Serum Albumin (BSA).
It may be necessary to check the appropriate antibody dilution.
If so, make 1/10, 1/100, 1/1,000 and 1/10,000 dilutions to
establish the correct titer. Working dilutions also may vary
with the manufacturer - check the literature
that accompanies your primary antibody.
- Place the coverslip in a petri plate containing filter
paper moistened with PBS. Make sure the cells are pointed up
when placed in the petri plate! Flood the coverslip with 50
ml of 1/40 dilution of the primary antibody (or as determined
in step 5).
- Incubate at room temperature for 1-4 hours. The incubation
may be left overnight if necessary.
- Wash 3X with PBS. Place coverslips in a new petri plate
containing PBS moistened filter paper.
- Apply 50 ml of FITC-labeled second antibody. A 1/100
dilution usually is satisfactory. You may need to determine
the appropriate dilution based on manufacturer directions or
through trial and error dilutions in the range of 1/10 to
- Incubate for 30 minutes at room temperature.
- Wash 3X with PBS.
- Place a drop of glycerin or appropriate commercial
fluorescent mounting media on a slide and place the coverslip
onto the slide with the cells facing down into the glycerin.
- Observe immediately with a fluorescent microscope
adjusted for fluorescein (490 nm excitation and 515 barrier
filter). The slides are best photographed using Kodak
Ektachrome or equivalent with an ASA or 200-400. An exposure
of 1-2 seconds is usually sufficient, although for low light,
30 seconds may be required. It is best to make a test exposure
roll if a photometer is not available.
Figure 2.7 Microtubules observed via fluorescent labeling
The procedure works for most primary antibodies merely by replacing
the anti-tubulin with another appropriate antibody (anti-actin,
anti-laminin, etc.). Just be sure to keep the secondary antibody
appropriate to the host for the primary.
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- email@example.com