BOT/MBIO/ZOOL 5364

THICK SECTIONING

Materials:

 

1.       Chuck (block holder) - round chuck for beam capsules flat chuck for flat embedding molds

2.       Ultramicrotome

3.       Sharp razor blades

4.       Specimen block

5.       Chuck support (tall for use with ultramicrotome; short for use with binocular microscope)

 

 

Procedure:

 

A.      Trimming Block

 

1.         Anchor specimen block in chuck and screw chuck into appropriate support.  Place under binocular microscope or in the ultramicrotome.

 

2.     Trim excess resin from the side of the block face.  Angle the cuts in order to provide a stable supported face (50˚) - pyramid shape.  An unstable face will vibrate at high frequency upon impact with the knife causing "chatter" (periodic variations in thickness).

 

3.     Trim excess resin from the top of the block face.  This is only necessary if the tissue is not at the block surface.  The more horizontal the cut, the less sectioning will be required to reach a full face.

 

4.     Trim the block face to form a broad trapezoid, the top edge being smaller than the bottom.  Avoid cutting away any tissue.  The block face may be large at this stage, but preferably less than 1 mm.

 

The advantages of the trapezoid block face are:

 

a.             Sectioning from wider to narrower means the sectioning is less wearing to the knife as the cut is made.

 

b.             Forms ribbons more easily if top and bottom are exactly parallel.

 

c.             Easy to orient direction of sectioning in the microscope.  The block will be in the microtome at the same angle each time it is sectioned (top is smaller).

 

d.           Provides orientation to the tissue.

 

If the top and bottom are not parallel, the block will not ribbon or the ribbon will curve.  This will also wear out the knives unevenly.

 

Use a new, clean razor blade for the final trimming.  This may also be done with a glass knife on a microtome.

 

a. A dirty or unsmooth lower edge will not contact the knife properly.

 

b. A dirty upper edge may cause the section not to detach from the block face and therefore be dragged back over the knife edge.

 

B.      Facing the Block

 

1.       Face the block using a glass knife (a boat does not need to be present and may interfere).  The microtome pivot control should be reset.  Advance the knife using the manual advance, but use the motor to move the specimen arm.  If the knife becomes dull, change knives.

 

2.       Reshape the block face into a trapezoid using a new, clean, sharp razor blade so that the top and bottom are parallel and the surface is glass-like.  There is a limitation of the width of the block as to available cutting area on the glass knife (approximately the left 1/2 of the knife is suitable for sectioning).  Larger sections are exceedingly difficult to obtain.

 

C.      Thick Sectioning

 

1.       Use a glass knife with a distilled water-filled boat using a syringe (be careful with the needle and keep it capped when not in use). Make sure pivot control is set appropriately (10 nm is our default setting).

 

2.       Cut thick section(s).  Remove sections from the boat with an empty slotted grid, clean wire loop or wooden spatula.

 

3.       Transfer the drop containing the thick section(s) to a drop of water on a clean glass slide.  Lift the transfer device away slowly.  The section should be floating in the drop.  Pretreating slides by dipping them in a warm solution of 0.5% gelatin in distilled water and letting them dry prior to use will make the drop “bead up” and permanently adhere the section to the slide.

 

4.       To accelerate adherence of the section, place the slide on a low temperature hot plate.  This will relax and flatten the section and evaporate water allowing attachment of the section(s) to the slide.  This may take approximately 2 minutes or so.

 

5.       Optionally, you may choose to stain the section if phase or other contrast microscopy are unavailable.  To stain the material, place a drop of stain (toluidine blue, or methylene blue) on section. Zoologists frequently use a mixture of methylene blue and basic fuchsin, called Paragon.

 

6.       Warm carefully until the edges of the drop begin to dry (30-60 sec).  Remove before boiling or the section will be folded.

 

7.       Remove from warmth; rinse gently with distilled water; dry.

 

8.       View with light microscope.  If you want to increase contrast, used phase or interference contrast microscopy or stain the material.  (Staining may be repeated to increase density.)

 

9.       It is possible to use oil immersion if desired.  For a temporary slide, mount cover slip with glycerol (removable with water).  For a permanent slide, seal the edges of the cover slip with epoxy glue.  ("Permount" causes the stain to bleach out over an extended period of time and is not recommended.)