BOT/MBIO/ZOOL 5364

 

THIN SECTIONING

 

1.    Use only freshly made knives.

 

2.    Choose desired area of block from thick sections and trim it down to about ½ mm wide (at bottom) X ¼ mm wide (at top) X ¼ mm high trapezoid.

 

3.    If you want serial thin sections, it is critical that the top and bottom edges of the trapezoid are perfectly parallel to each other.

 

4.    Check to see that pivot control is set to the thin sectioning thickness (10 nm).

 

5.    For initial approach of the knife to the block in the ultramicrotome, use the center portion of the knife.  Once you have faced off the block, you can move to the sharper left edge.

 

6.    Use the shadow on the block to determine the distance of the knife from the block.  A small strip of tinfoil held behind the knife can aid in seeing the negative image of the shadow.  Advance the knife until the shadow disappears, then turn on the motor and wait until the block is sectioned; you may advance the knife with the fine advance knob with extreme caution.

 

Note:      Only change motor speed when motor is on.

 

7.       Make tilt (block) and lateral (knife) adjustments if necessary.  The best vertical angle is 90° to the direction of sectioning.  Horizontal angle is less critical.  Be sure that locking clamps are relaxed and then tightened “spring-tight”. 

 

8.       Pick up sections on clean grids* and stain if necessary.  You may wish to delay staining for several hours if your sections tend to fall off during the staining process.

 

 

*to clean grids, sonicate them for a few minutes each in a solution of:

 

a)       dilute hydrochloric acid (1N, for instance) in a 1:1 ratio with

 

b)       95% ethanol