| Contributor: |
The Laboratory of Rebecca Heald in collaboration with John Merlie, Jr. at the University of California, Berkeley
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| This protocol describes an in vitro reaction to assay mitotic spindle assembly. The assay uses Cytostatic Factor extract made from Xenopus eggs, fluorescently-labelled tubulin, and prepared sperm nuclei. Spindle assembly is monitored by immunofluorescence microscopy. |
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1. On ice, add 0.5 μl of Rhodamine-labeled tubulin and 2.5 μl of 20X Sperm Nuclei to 50 μl of Cytostatic Factor (CSF) Extract (see Protocol on Labeling Tubulin with Fluorescent Dyes, Protocol on Sperm Nuclei Preparation, and Protocol on Cytostatic Factor (CSF) Extract Preparation for Spindle Assembly) in a 1.5 ml tube (about 100 sperm per μl of extract).
2. Incubate for 10 min at 20°C.
3. Release the extract into the interphase by the addition of 5 μl of Calcium Solution. Mix the sample by pipetting up and down using a cut off pipette tip.
4. Incubate for 80 min at 20°C.
5. Check that the extract is in the interphase by transferring 1.2 μl to a microscope slide using a cut off pipette tip. Carefully place 6 μl of Spindle Fix on top of the drop of extract and squash gently by placing a 22 x 22 mm coverslip on top. If the sample is to be saved, seal the coverslip to the slide with clear nail polish. Under fluorescence microscopy, nuclei should appear large, round, and uniform. Microtubules should be long and abundant.
6. At 90 min post Calcium Solution addition, add 0.5 volume (25 ul) of fresh CSF Extract to the reaction. Continue the incubation at 20°C.
7. Take samples at 15, 30, 45, 60, and 90 min to assess the spindle assembly reaction.
8. For immunofluorescent analysis of samples, transfer 10 to 20 μl of spindle assembly reaction to a 1.5 ml microcentrifuge tube and add 1 ml of Dilution Buffer.
9. Layer this mixture over 5 ml of Cushion Solution in a 15 ml modified Corex tube containing a 12 mm2 coverslip (see Hint #2).
10. Centrifuge the tubes at 16,000 X g (10,000 rpm using an HB-4 rotor) for 15 min.
11. Aspirate the supernatant and Cushion Solution before removing the coverslip.
12. Post-fix the coverslips in Methanol at -20°C for 5 min.
13. Transfer the coverslips to PBS and stain with the Primary Antibody and Secondary Antibody (see Protocol on Immunofluorescence and Hint #3).
14. The DNA is stained with Hoechst Dye Solution for 2 min.
15. Wash the coverslips in PBS and place them upside down on a 4 μl drop of Mounting Medium and seal the slide with nail polish.
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| 20X Sperm Nuclei |
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| Mounting Media |
| 90% (v/v) Glycerol
10% (v/v) 0.2 M Tris-Cl, pH 8.0
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| 0.2 M Tris-Cl, pH 8.0 |
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| BRB80 (5X) |
| 4 M PIPES
Add the salts to ddH2O and add solid KOH pellets until the PIPES dissolves
Adjust the pH to 6.8 with 10 M KOH
5 mM MgCl2
5 mM EGTA
pH 6.8
Filter sterilize and store at 4°C
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| Cushion Solution |
| 40% (v/v) Glycerol
Prepared in 1X BRB80
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| 10 M KOH |
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| Dilution Buffer |
| Prepared in1X BRB80
1% (v/v) Triton X-100
30% (v/v) Glycerol
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| Hoechst Dye Solution |
| 10 mg/ml Bisbenzimide (Hoechst 33258)
Store in the dark at 4°C
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| MMR (20X) |
| 100 mM HEPES, pH 7.8
Store at room temperature
40 mM KCl
40 mM CaCl2
20 mM MgCl2
2 M NaCl
2 mM EDTA
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| Spindle Fix (1 ml) |
| 100 μl of 10X MMR
0.5 μl of Hoechst Dye Solution
600 μl of 80% (v/v) Glycerol
300 μl of 37% (v/v) Formaldehyde (commercial stock) (CAUTION see Hint #1)
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| Calcium Solution |
| 100 mM KCl
1 mM MgCl2
Store in aliquots at -20°C
4 mM CaCl2
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| 400 mM CaCl2 |
| Store in aliquots at -20°C
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Rhodamine-Labeled Tubulin
HEPES
EDTA
Potassium Chloride
Sodium Chloride
Methanol
Formaldehyde
EGTA
Triton X-100
PIPES
Calcium Chloride
Magnesium Chloride
Glycerol
Primary Antibody
Potassium Hydroxide
Secondary Antibody
Tris
Hoechst 33258
Nail Polish
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. The 15-ml glass Corex tubes are modified in the following way. The rounded bottom of the tube is filled with silicone. On top of the silicone is a fixed piece of plexiglass. Resting on the fixed section of plexiglass is a plexiglass insert. The coverslip rests on the plexiglass insert. A thin metal rod with a hook at the end is used to retrieve the coverslip from the centrifuge tube. (See Evans L, Mitchison T, Kirschner M. Influence of the centrosome on the structure of nucleated microtubules. J Cell Biol. 1985; 100:1185-91.) for a diagram of the modified tube and the tool.
3. Frog extracts have huge pools of biotin and it is generally a good idea to avoid avidin-biotin detection systems.
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