PHYSICAL FIXATION

A. Quick freezing- It is most important that the tissue be frozen quickly, preserving enzyme activity and antigenicity as close to in-vivo as possible. Freeze artifacts may be introduced during the freezing process if the sample is frozen too slowly. The ice crystal formation size and amount is directly proportional to the speed of freezing. These artifacts can be seen as holes when thawed and are quite evident microscopically.

1. Cryogens- There are many methods of freezing sample for cryosectioning. Often the tissues are frozed in the cryostat itself. A precooled isopentane bath is also used routinely. My preference for freezing tissue (surrounded by a commercial embedding compound) is placed directly into liquid nitrogen. LN2 is about -195 degrees Celsius and will cause a gas bubble to form if tissue is placed directly into it. This gas layer will impede freezing causing artifacts if not surrounded by media.

2. Cryoprotectants- To aid in the assurance that ice crystals do not form, a cryoprotectant is sometimes employed. Some common ones are 25-30% sucrose, glycerol and PVP.

B. Cutting frozen sections is not difficult, but the following recommendations make for a more satisfactory product.

1. Keep the microtome clean and lubricated.

2. Knife must be sharp, clean and nick-free.

3. Knife and chuck must be secure and tight.

4. Clean your knife between sections.

5. Temperature must be appropriate for the type of tissues you are cutting.

The anti-roll plate is a swing away plastic plate that rests against the knife. It must properly be adjusted to function correctly. Most need to be parallel to the knife edge. The temperature of everything is important in the cryostat. Touching the knife, the block, or the anti-roll plate will warm them. If the anti-roll plate is not touching the knife it will warm slightly and cause the sections to stick.

Tissue Processing- Once the tissue is fixed it must be surrounded and infiltrated with a matrix to increase stability for sectioning.

Dehydration - Tissues in aqueous solutions cannot usually be infiltrated with most medias. Water removal must be achieved gradually through a series of graded dehydrant (alcohol or acetone). Typically 50% 70% 95% 100% solutions are used.

At the microscopic level, forces between the liquid/gas interface will cause enough pressure to distort a biological sample, therefore it is not a good idea to let samples dry out during processing.

Clearing- After dehydration samples can be placed in a nonaqueous liquid miscible with the embedment media.

1. Xylenes are the most common clearing agent for paraffin embedment.

2. Toludine is more tolerant of residual water, but is 3x more expensive.

3. Chloroform is slow and a health hazard.

4. Methyl salicylate is expensive, but smells nice (also known as oil of wintergreen).

5. Some newer xylene substitutes are available and a must if sectioning beta-galactosidase (beta-gal) stained tissue. Most of these are limolene based. Limolene is a volatile oil found in citrus peels (smells wonderful). They are less of a helath hazard and I have found them to be quite good, as long as the tissues are well-fixed.

Embedment media- In order to cut biological tissue very thinly (to send photons or electron through), it must be embedded in a hard substance for support. The thinner the sample needs to be, the harder the embedment media must be. Infiltration of the media in it's liquid form is crucial to good sectioning. A vacuum can sometimes be applied for difficult tissues.

1. Paraffin is the most common media and can be purchased for use at different melting points.

2. Plastics are also available for thinner sections or special staining.