Preparation of yeast cells for immuno-electron microscopy.
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Used by Prof. Chuck Cole's lab at DMS.
A) FIXATION WITH ADDED OSMOTIC SUPPORT
1) Grow cells to early log phase (OD600 of 0.5 - 0.6) in YEPD or SD medium.
Typically we process 100ml per sample.
2) Quickly harvest cells over a disposable 0.45um filter unit with a vacuum of 15-20 psi. Swirl while filtering to a final volume of 5ml. Disconnect from the vacuum and add 25ml of fixative directly to the cells. Quickly swirl to cover cells, and use a 10ml disposable pipet to resuspend the cells by pipetting up and down.
3) Transfer the cell suspension to 50ml conical tubes and incubate on ice 30 minutes.
Fixative: 0.04M Potassium phosphate buffer (pH6.7)
Try 0.25M - 1.25M Sorbitol
3% Formaldehyde (16% methanol free, Polysciences, Snap vial)
Try 0.25 to - 0.5% Glutaradehyde (EM grade, Polysciences, Snap vial)
0.05% 1mM MgCl2
1mMCaCl2
Fixative is made within 1 hr. of processing cultures and held on ice in 25ml aliquotes in 50ml polypropylene conical tube (Corning). Tubes can be reused at step 3.
Filtering and transferring cells to ice should take 2-3 minutes.
B) SODIUM METAPERIODATE TREATMENT
4) Pellet cells in a clinical centrifuge (IEC on setting 7 for 3 minutes or in Sorval at 3-4 k rpm, SS34 rotor for 5 minutes) at room temperature (RT) - avoid pelleting the cells too densely. Pour off supernatant.
5) Resuspend and wash cells in 25ml of the following solutions. Pellet cells at RT as above:
1X - 0.75M sorbitol in 0.04M potassium phosphate buffer (pH 6.7)
1X - 0.25M sorbitol in 0.04M potassium phosphate buffer (pH 6.7)
1X - 0.04M potassium phosphate buffer (no sorbitol) (pH 6.7)
6) Pellet and Resuspend cells in 10ml of 1% sodium metaperiodate (NaIO4). Make solution just prior to incubation
Incubate 15 minutes at RT.
Cell pellets can easily be resuspended by gently mixing with a wooden dowel (ASP# A5000-1). Cell clumps should be 1mm..
C) AMMONIUM CHLORIDE TREATMENT
7) Pellet and wash cells at RT.
1X ddH20 or Milli-Q H20 (25ml).
8) Pellet and resuspend cells in 10ml of 50mM ammonium chloride (NH4Cl).
Incubate 15 minutes RT.
D) DEHYDRATION
9) Pellet and wash cells at RT.
1X in 20ml ddH20 or Milli-Q H20
1X in 5ml ddH20 or Milli-Q H20 - transfer cells to disposable glass test tubes (13 x 100mm) with a Pasteur pipet prior to pelleting.
10) Pellet and resuspend cells in ice-cold, ethanol H20 series: (5ml each)
1X - 50% EtOH
1X - 70% EtOH
1X - 80% EtOH
1X - 90% EtOH
1X - 95% EtOH
2X - 100% EtOH - use a fresh, unopened bottle of EtOH
1x - 100% EtOH - this step at RT, with RT EtOH
Dehydrations should be done for 5 minutes/step and on ice with ice cold ethanol (except at final 100% step as noted above). Centrifugations should also be done at 40C during dehydrations.
Dehydrations should be done as quickly aspossible (definitely no over-night i ncubations).
Do not pellet cells too densely. Pellet should be a little loose; try 2' in IEC clinical on next to the highest setting (6). Again use wooden dowels to resuspend cells in EtOH.
E) INFILTRATION
11) Pellet cells and pour off EtOH.
Resuspend in 2ml of a 2:1 mixture of EtOH: LR White resin
(Polysciences)
Place on a roller for 1 hr. at RT.
12) Pellet cells and pour off EtOH/resin.
Resuspend in 2ml of 1:1 EtOH: LR White resin.
Place on roller over night at RT.
13) Pellet cells and pour off EtOH/resin.
Resuspend in 1ml of 1:2 EtOH: LR White resin.
Place on roller 1 hr. at RT.
14) Pellet cells and aspirate resin with a Pasteur pipet.
Resuspend in 1ml 100% LR White resin.
Place under a 15 - 20 psi vacuum for 15' at RT.
LR White resin and EtOH should be at RT during infiltrations.
Again use dowels to resuspend cell pellets, clumps should be 1mm.
F) EMBEDDING
16) Remove samples from the vacuum and transfer cells and resin with a Pasteur pipet to gelatin capsules (Ted Pella), capsules should be about 1/2 full. A 100ml culture at an OD600 of 0.6 should produce enough material for 3 capsules.
17) Gently pellet the cells in the capsules by briefly (1 min. on the highest setting) centrifuging in a swinging bucket. An adaptor that holds the gelatin capsules upright will be necessary. We use an IEC Centra - 4B centrifuge with swinging bucket adaptors (Ultrac cat. #7226, tube size 12 X 75mm) into which has been placed P1000 pipet tips, cut to a height which leaves the top of the gelatin capsules flush with the top of the bucket adaptors.
18) Top off the capsules with fresh LR White resin, add labels and gelatin caps. Place capsules in a heat block set at 47°C. Cover heat block with lead or several layers of aluminum foil to keep the temperature stable. Polymerize for 2 days at 47°C.
We strongly recommend the papers by R. Wright and J. Rine, Methods in Cell Biology, (1989) 31:473-512, and by E. van Tuinen and H. Riezman, J. Histochem. and Cytochem., (1987) 35:327-333.
A detailed protocol from Robin Wright on which the above is based is here.