レプリカ作成法

Preparing for platinum replicas of MSP fibers for TEM

This method is adapted from a correlative light and electron microscopy paper  (Svitkina and Borisy, 1998)  and concentrates only on preparation of MSP fibers for platinum coating.  

    Before starting preparation make sure to incubate 100 % ethanol with molecular sieves for a few days to get rid of water.

Fixing fiber;  The biggest problem with fixing fibers from S100 for electron microscopy is the high concentration of protein found in cell extracts (> 100 mg/ml).  This can be eliminated from the preparation in one of two ways.

a)      Dilute S100 to 1:30 for growing fibers

b)      include a washing step after fiber growth and prior to glutaraldehyde fixation. 

From my experiences the second method works better.   Because fibers are stickier with higher concentration of S100 and during dehydration some fractions of fiber are lost.   Therefore more fibers remain on coverslips with higher S100 concentration. 

 Dehydration

1.  prepare small coverslips by cutting a 22 x 22 mm coverslip into three 7 x 22 mm pieces, and then, cut each pieces into one-third to make rectangle-shaped pieces.  Cut off right-upper corner of them so that you can tell which side you put your sample later.  

2.  1:5 dilution of S100 in KPM with 1 mM ATP is prepared and 10 ul is pipetted on to coverslips. Incubate them in a moist chamber to prevent them from drying

3.  After incubation of desired time,  hold the edge of coverslips with a forcep and dunk them into KPM, then put on a droplet of 2.5 % glularaldehyde (in KPM) for 20 min at room temperature. 

    For Immunogold-labeling go to Section II after this step.   

4. Incubate coverslips in freshly prepared 1 mg/ml Tannic acid in H2O for 20 min.  No washing is necessary before this step.  I usually use 24-well plate for step 1-3.  

5. Rinse coverslips twice by dunking them into distilled water, and incubate them in distilled water for 5 min.  Be sure to remove tannic acid completely.   Tannic acid makes precipitation with uranyl acetate , which ruins your samples. 

6. Rinse coverslips again with distilled water and then incubate them in 2 mg/ml uranyl acetate in distilled water for 20 min at room temperature.  Filter uranyl acetate solution before use it. 

7. Wash coverslips with distilled water and load them with sample side up into a wire basked alternating them with pieces of lens tissue fitting the size of the basket.

8. Transfer the basket through a series of graded ethanols; 10, 20 40, 60, 80 % and 100 % twice, each for 5 min. Transfer the basket quickly from a beaker to a beaker to avoid drying.  

9. Transfer the basked into 2 % uranyl acetate in 100 % EtOH and incubate it for 20 min. Filter UA before use

10. Incubate the basket with 100 % EtOH twice, followed by 100 % EtOH (dried over molecular sieve) wash twice. 

11. CPD – 10 to 12 change of CO2.  Make sure that the liquid always covers your sample. 

12. Platinum and carbon coating using a vacuum evaporator.  If dried samples are not coated right after CPD, they should be kept in a desiccator to avoid moisture.  

Coating

 I always use 10 mg platinum for coating.  The distance between a sample and platinum is 10 cm and the angle is 45 deg.  

Mounting on grids

• Prepare copper grids with 0.25 % formvar

• In a 3x4 tissue culture dish, prepare one well of 20 % Hydrofluoric acid in dH2O.  Add water to two more wells to prepare for washing.  To the second well, add 5 ul of 0.01 % Triton X-100 to decrease surface tension.  Also, use the same the same dH2O for all wells to prevent temperature differences. CAUTION: Hydrofluoric acid is extremely dangerous. 

• Score the replica with a razor blade into small pieces.  Use a new blade and if necessary do this under a dissecting microscope. 

• Float a converslip with replica side up into the well containing Hydrofluoric acid.  Wait for the converslip falling off from the replica.  Pieces of the replica will break apart along the razor blade scores. Do not force the converslip to fall off, this could break the replica.  

• With a platinum loop, gently pick up each replica and transfer to the well with dH2O with Triton X-100 and wait one minute.  Finally transfer the replica to the dH2O well.

• Replicas can be picked up with grids by dunking the grid.  Gently, with the formver-side up, pick up a replica from underneath.  If you do tomography you sample must be on dead center, using a dissecting microscope is recommended. 

• Wait for the grid to dry and put it in grid box.