Download Immunofluorescence

Background Information
Drug Treatments
Cells will be treated with three different drugs prior to lab. These are the three different drugs that
Pat was using. When they are initially purified and tested, potential drugs are often given an
alphanumerical code because naming useless compounds is a waste of time. Pat’s compounds are
designated CD16, OL32, and PT08.
Pat’s observations after two days of drug treatment:
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CD16: Most cells were binucleate or multinucleate.
OL32: Most cells have single nuclei that are oversized with too much DNA.
PT08: Most cells have aneuploid nuclei or nuclei that are oversized.
The concentration of drug in your treatment and the length of the treatment time will vary. So these
are important pieces of information to record.
CHSE-214 Cells
We will be using CHSE-214 cells, which are a cultured line of cells isolated from Chinook salmon
embryos (Oncorhynchus tshawytscha). The advantage to using fish cells over mammalian cells is
that the CHSE-214 cells grow well at 21 (room temperature) and that they do not require an
environment supplemented with CO2 as many mammalian cells do. These cells also divide rapidly.
In addition, a search of the literature on CHSE-214 cells revealed that no one has published any
information on the distribution of microtubules and actin filaments in these cells. Therefore, you
will be the first ones to do these experiments.
These embryonic cells are probably not fully differentiated (specialized), but they do exhibit a
general epithelial morphology. Under the culture conditions we are using here, epithelial cells do
not necessarily form clear apical and basolateral polarity. No data exists as to whether CHSE-214
cells are polar under these growth conditions.
Cytoskeleton
All eukaryotic cells have a cytoskeleton comprised of two different elements: microtubules and
actin filaments.1 The cytoskeleton forms a framework which supports the cell, determines the
overall shape and organization of the cell, and serves as tracks for the generation of motility. In
general, the functions of the microtubules and actin filaments in a particular cell are distinct.
However, the two systems interact and often have overlapping or complementary functions.
One case where the functions of microtubules and actin filaments are intertwined is in animal cell
division. The mitotic spindle is made of microtubules, and the chromosomes move on microtubules
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Many animal cells also possess a third cytoskeletal component, intermediate filaments. You can read more about the
cytoskeleton in Chapter 9 of your textbook.
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during mitosis. The actin filaments are responsible for dividing of the cytoplasm, called
cytokinesis.
Microtubules are long, tube-like polymers built from -tubulin and -tubulin proteins.
Microtubules usually initially grow from a single spot (or in some cases a few spots) called a
microtubule-organizing center (MTOC). One end of the microtubules usually remains anchored in
the MTOC.
Actin filaments are long, string-like polymers built from actin proteins. Actin polymerization
occurs at numerous sites throughout the cell. The organization of actin filaments appears less
organized to our eyes, but it is precisely controlled in the cell.
Fluorescent Labeling of Cell Structures
We will use indirect immunofluorescence to label microtubules. Microtubules will appear
green. The following two antibodies will be used for this:
1º antibody: mouse anti--tubulin
2º antibody: FITC-sheep anti-mouse IgG
We will use Texas Red phalloidin to label actin filaments. Phalloidin is a fungal toxin produced by
the mushroom Amanita phalloides. Phalloidin specifically binds to filamentous actin. Binding is
essentially irreversible. The Texas Red fluorochrome is covalently attached to phalloidin. Actin
filaments will be red.
The DNA will be labeled with DAPI (4’,6-diamidino-2-phenylindole). DAPI is a chemical that
naturally fluoresces and intercalates between the bases in double-stranded DNA. The DNA will
appear blue.
Because antibodies are not involved in labeling the actin or DNA, these labeling techniques are not
called “immunofluorescence.” These two methods are simply called fluorescent labeling.
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Indirect immunofluorescence protocol for labeling the microtubules,
actin filaments, and DNA in tissue culture cells
WARNINGS:
Be sure to keep track of which coverslips are drug-treated and which have control cells.
Be sure to keep track of which side of the coverslip your cells are located on.
There is no way to determine treatment or cell side if you lose track.
Do not let the coverslips dry out at any time during the procedure.
Drug Used___________________
Drug Concentration _________________
Length of Treatment_____________________
1. Observe the living control and treated CHSE-214 cells in phase contrast with an inverted
microscope.
2. Pipette out the culture media and replace with PBS (Phosphate Buffered Saline).
3. When you have a tube of fixative at your bench, remove PBS and thoroughly coat coverslips
with fixative. Fix for 15 min.
4. While cells are fixing, add PEMK + Triton X-100 to a 25 mm diameter plastic top (serving as a
cup here) just above the ridges (1/2 to 2/3 full or ~12 ml).
5. Using forceps, remove coverslips from fix, touch one edge to a Kimwipe to dab off excess fix.
6. Wash cells by placing the coverslips in the PEMK-Triton cup so that the cells are up. Incubate
(wash) for 2 min.
7. Dry the back of the coverslips briefly on a flat towel and place it (face up) on the supports in the
humid chamber. Coat each coverslip with 75 l of PEMK-Triton-BSA, labeled "BSA". Cover
and incubate for 10 min.
8. During the 10 minute incubation, get three clean cups and add PBS to all three.
9. Wash the coverslip in one PBS cup for 2 min.
10. Transfer the coverslip to another PBS cup and wash for 2 min.
11. Repeat Step 10 one time. (This makes a total of three washes.)
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12. Dab off excess PBS, and dry the back of the coverslip briefly on a flat towel. However, do not
let the top of the coverslip dry out thoroughly.
13. Place coverslip (face up) on supports in humid chamber.
14. Turn out overhead lights.
15. Coat coverslip with 75 l of primary antibody solution (= mouse anti--tubulin, TR-phalloidin,
or both). Cover the humid chamber and incubate for 30 min. Be sure to keep this antibody in
the dark. To prevent exposure to light, turn down room lights if possible while handling the
coverslips and cover your humid chamber with a box.
16. During the 30 min incubation, wash out your cups. Then refill them with fresh PBS.
17. After the 30 min incubation, dab the edge of the coverslip to remove excess antibody. Wash
coverslips in PBS, 3 times, 2 min each wash.
18. After the third wash, dab off excess PBS, and dry the back of the coverslip briefly on filter
paper. Again, do not let the cells dry out.
19. Place coverslip on support in humid chamber.
20. Coat coverslip with 75 l of secondary antibody solution (FITC-sheep anti-mouse IgG). In
addition, the secondary antibody solution will contain DAPI, which stains nucleic acid and
highlights the nucleus. Incubate for 30 min. Be sure to keep this antibody in the dark. To
prevent exposure to light, turn down room lights if possible while handling the coverslips and
cover your humid chamber with a box.
21. During 30 min incubation, wash out your cups.
22. Dab the edge of the coverslip to remove excess antibody. Wash coverslips in PBS, 3 times, 2
min each wash.
23. After the third wash, dab off excess PBS, and dry the back of the coverslip briefly on filter
paper.
24. Place a small drop of mounting medium on a clean slide. Place the coverslip with the cell side
down onto the slide.
25. If excess mounting media leaks out from around the edges of the coverslip, dry off with a filter
paper triangle.
26. Coat edges of the coverslip with nail polish to form a water-tight seal between the coverslip and
the slide. This will likely require two coatings, with a brief time between coats for drying of the
first coat.
27. Label your slide with your name and the type and length of treatment.
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28. Place slides in a light-tight box for drying. The instructors will place them in the refrigerator or
at -20º C later.
References:
Lannan CN et al. 1984. Fish cell lines: establishment and characterization of nine cell lines from
salmonids. In Vitro 20: 671-676.
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