Yeast Meiotic Spreads for Electron Microscopy

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Tony Rook
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Yeast Meiotic Spreads for Electron Microscopy

Please find the following sample prep protocol:

Yeast Meiotic Spreads for Electron Microscopy

Procedure:

A. Prepare Plastic Coated Slides

1. Wash slides with appropriate slide detergent solution.

2. Wash slides in ddH2O.

3. Dip the slides in a solution of 95% Ethanol and wipe dry with lens paper.

4. Dip slides in Plastic Solution for approximately 2 seconds and remove rapidly and vertically.

5. Check thickness of plastic. Under phase contrast, slides should have few (if any) bubbles in the plastic coat.
A quick and accurate way of checking the plastic thickness is to gently float plastic coated slides in water. Plastic will come off and have a bronze reflection. A silver reflection is too thin and a purple reflection is too thick.

B. Preparation of Cells and Mounting of Cells

1. Sporulation occurs at approximately 5 hours for SK1 derivatives and at approximately 14 hours for BR derivatives.

2. Decant the supernatant and resuspend the cells in 1 ml of 2% Acetate Solution and incubate at room temperature for 10 min.

3. To the solution containing the cells add 10X Zymolyase 100T to a final concentration of 25 μg/ml. You can increase the final concentration of Zymolyase 2 to 3 fold depending on your specific conditions (this should be determined empirically).

4. Incubate the cells at 30°C until 80% of the cells are sphereoplasted. A rapid way of estimating sphereoplasted is to add 5ul of cell suspension to a standard microscope slide. Combine with 2 μl of 10% (w/v) SDS solution. Cover with a cover slip and estimate how many cells lysis.

5. Centrifuge to pellet the cells (approximately 1,500 X g) and discard the supernatant.

6. To the cell pellet add 1 ml of ice-cold Wash Solution and mix well by inversion.

7. Centrifuge to pellet the cells and discard the supernatant.

Work quickly for Steps #8, #9 and #10.

8. Resuspend the cells in 100 μl of Sulfonic Acid Solution and mix with a pipette tip.

9. Add 490 μl Paraformaldehyde Solution.

10. Add cellular suspension to slide and allow to stand at room temperature for 10 to 30 min (with plastic slides make sure the pipette tip does not touch the slides).

11. Drain excess liquid from slide and add 350 μl of Paraformaldehyde solution and allow to stand at room temperature for 5 min.

12. Rinse gently with 5 ml of 0.4% Photoflo and air dry.

13. Mix 300 μl of Stain Solution and 150 μl Gelatin Fix solution in a microcentrifuge tube.

14. Immediately pipette the 450 μl solution onto a slide at 60°C (in a slide warmer).

15. Wait for approximately 2 min or until the stain appears brownish.

16. Rinse off solution using ddH2O and allow to air dry.

17. Score the area around cells with a razor blade and drop slide into ddH2O bath at room temperature.

18. Collect plastic mounts with cells using 50 mesh copper grids.

Solutions:

Gelatin Fix
9.9 ml ddH2O
110 μl Formic Acid
0.2 g Gelatin

Stain Solution
Filtered to remove insoluble material
50% (w/v) Silver Nitrate (CAUTION Biohazard!)

Photoflo
0.4% (v/v) Photoflo

Sulfonic Acid Solution
0.1 M MES
0.5 mM MgCl2
1 mM EDTA
pH 6.4 using KOH

Wash Solution
0.1 M MES
0.5 mM MgCl2
1 mM EDTA
1 M Sorbitol
pH 6.4 using KOH

Paraformaldehyde Solution
Add 10 μl of 1 M NaOH to 50 ml and heat,
covered with tin foil until dissolved
Prepare just before use and let stand on ice
4% (w/v) Paraformaldehyde, pH 7.0 (CAUTION Biohazard!)

Zymolyase 100T (10X)
2.5 mg/ml Zymolyase 100T

2% Acetate Solution
10 mM DTT - Add just before use
0.8 M Sorbitol
2% (w/v) Potassium Acetate
pH 7.0

Plastic Solution
Stir for 20 min and filter to remove un-dissolved plastic
Dissolve in Chloroform (CAUTION Biohazard!)
0.5% (w/v) Falcon Optilux Petri-Dish (brand is important)

95% (v/v) Ethanol

1 M HCl

Bioreagents and Chemicals:

Zymolyase 100T
Paraformaldehyde
Magnesium Chloride
Hydrochloric Acid
Chloroform
Sodium Hydroxide
Sorbitol
Gelatin
MES
DTT
Silver Nitrate
Potassium Hydroxide
Potassium Acetate
EDTA
Ethanol
Photoflo
Formic Acid

Reference Link:

http://www.bio.com/protocolstools/protocol.jhtml?id=p203