Sectioning or Microtomy

Microtomy or sectioning

Preparation of paraffin tissue block for sectioning

1- Trimming the face of the paraffin tissue block - Before inserting the paraffin tissue block into the

microtom’s chuck, be certain that the upper and lower edges of the block are parallel to each other and to the knife edge, otherwise ribboning of the tissue sections will not occur, or if occur, a straight ribbon will not be obtained.

- Trim the face of the block with heated scalpel. If there is excess paraffin on the face of the tissue block, trim the block, leaving just a few millimeters of paraffin surrounding the tissue.

- An excess of paraffin can cause wrinkling, tissue deformation and prevent flattening of the sections when sections are spread.

Preparation of paraffin tissue block fro sectioning

2- Attachment of paraffin tissue block to object carrier.

- If the mold that was used for embedding is removed after the paraffin solidifies, the paraffin block must be fused to an object carrier of either metal or wood by paraffin.

- Usually the face of the object carrier is grooved to increase surface contact between the paraffin block and the tissue carrier.

Preparation of paraffin tissue block fro sectioning

Microtomy or sectioning

• Microtome is an instrument used for sectioning paraffin-embedded tissues for light microscopy. The rotary microtome is most widely used to cut thin sections of paraffin infiltrated tissues.

• The hard blocks containing the tissues are then taken to a microtome and are sectioned by the microtome's steel to a thickness of 1–10 µm.

• Remember that 1 micrometer (1 µm) = 0.001 mm = 10–6 m; 1 nanometer (1 nm) = 0.001 µm = 10–6 mm = 10–9 m.

- Rotation of the drive wheel moves the tissue-block holder up and down. Each turn of the drive wheel advances the specimen holder a controlled distance, generally between 1 and 10 micrometers. - After each forward move, the tissue block passes over the knife edge, which cuts the sections.

Microtome Knife

• The microtome knife is made of high grade stainless steel.

• It has its own fitted back and handle to use when manually sharpening the knife.

• The sections are floated on water and transferred to glass slides to be stained.

Microtomy or sectioning…Con

• A completely different way to prepare tissue sections is to submit the tissues to rapid freezing.

• In this process, the tissues are fixed by freezing (physically, not chemically) and at the same time become hard and thus ready to be sectioned.

Microtomy or sectioning…Con

• A freezing microtome—the cryostat (Gr. kryos, cold, + statos, standing)—has been devised to section the frozen tissues.

• Because this method allows stained sections to be prepared rapidly (within a few minutes), it is routinely used in hospitals to study specimens during surgical procedures.

Microtomy or sectioning…Con

• Freezing of tissues is also effective in the histochemical study of very sensitive enzymes or small molecules, since freezing does not inactivate most enzymes.

• Because immersion of tissues in solvents such as xylene dissolves the tissue lipids, the use of frozen sections is advised when these compounds are to be studied.

Microtomy or sectioning…Con

The cryostat

1- Check out the Microtome, Clean, oiled, Chuck mount cranked back, clean Knife properly installed. 2- Set thickness of sections at 10 microns. When you have more skill the thickness may be reduced. Thinner sections will allow a more detailed study of the tissue. 3- Clamp the block in the microtome carrier so that the upper and lower surfaces are parallel with the knife edge. This will be demonstrated. Do not change the angle of the blade. Move the blade toward the block so that it just clears the block and LOCK the blade so it does not move.

Preparation of the Microtome for Sectioning

During sectioning, the sections become compressed and wrinkled and have to be flattened before they firmly fixed to the slides.The spreading of sections takes place by floating the ribbons of tissue sections on warm distilled water at a temperature about 5-10 ° C below the melting point of the paraffin.So water temperature of 45-50◦C will soften the paraffin to spread the sections and remove the wrinkles.The temperature should not melt the paraffin as this can cause shrinkage, tearing and displacement of tissue components.

Spreading the sections

attachment and Mounting of sections to glass slides

- If temperature is not warm enough, tissue will not flatten properly.

- The tissue must be attached to a solid surface for support. So the tissues are attached to glass slides which give them support.

- Use perfectly clean glass slides, label it with code number of the tissue that will be attached to this slide.Center the section on the slide