Tissue Preparation for Fixation
Tissue specimens of interest should be placed in a fixative of choice. The volume of fixative should amount to, at the very least, 10x the size of the specimen. There should be ample space surrounding the specimen so that proper infiltration of the fixative can occur. If fixation is incomplete or takes too long to occur, the finished product will not show the correct detail under the microscope. Improper fixation cannot be compensated for later in processing.
Choice of Fixatives
The most common fixative is commercially bought 10% buffered formalin. It is always available in the histology lab and is a good general fixative that works well with most stains. It is often preferable, when doing immunohistochemistry, to use freshly prepared 4% paraformaldehyde PARAFORMALDEHYDE/PBS.
1. Measure 100ml Phosphate Buffered Saline (PBS) into a measuring cylinder. Pour into the conical flask containing 4g of paraformaldehyde. Cover with parafilm and transfer to the fume hood: thoroughly shake - take care not to splash paraformaldehyde about - it is a rapid fixer and is TOXIC.
2. Place flask on top of the hotplate/stirrer inside the fume cupboard and set the heat control to 7 with moderate stirring. Allow the solution to warm up-it will turn from being cloudy to clear when ready. Inspect regularly to avoid over-heating and consequent spilling.
3. When the paraformaldehyde has dissolved, switch off the heat but leave to stir: do not handle for safety reasons. Allow to cool.
4. When cooled, transfer the fixative to a 4C refrigerator. Label appropriately and date. The reason for this is that the commercial product contains methanol which is used as a preservative. It is possible that the methanol masks some epitopes, thus reducing the success of some immuno staining. A prolonged time in the formalin or paraformaledhyde causes cross-linking of protein, also obfuscating antigenic sites. A specimen should not be in the fixative for more than 4 hours. Therefore, it is essential that the sample to be processed be no thicker than 3mm otherwise infiltration will be incomplete in this time. Very tiny specimens can be left in the fixative for shorter periods of time 1 hour is often sufficient. Paraformaldehye should be prepared by the submitting lab. Concentrations can vary according to type of tissue and how sensitive the antigen in question is to cross-linking.
Histochoice is another fixative available in the histology lab. This is purchased from Amresco and contains no formaldehyde. It reduces the possibility of cross-linking and is often desirable for immuno staining. Also, Prefer is available from Anatech.
Bouins fixative, also available in the histology lab, is an excellent nuclear stain. It does not necessarily work for immnohistochemistry and must be used with caution. There are many other fixatives for specialized procedures and the lab should be consulted if any of these should be required.
If specimen contains bone, it must be decalcified. Generally this is done in the histology lab using a mild commercial preparation. There are many decalcifying agents; most contain acid of some sort. If the specimen is to be kept for awhile before processing, it should be transferred out of the fixative and put into 70% ethanol. This is a good storage medium in which tissue can be stored without loss of antigenicity and without incurring too much shrinkage.
This consists of passing the tissue samples through a series of ethanols up to absolute, then into xylene or xylene substitute and finally into paraffin. It is very important that the tissue has been fixed properly, otherwise complete dehydration cannot happen. If not completely free of water, the tissue will not clear completely and will not infiltrate with paraffin to the extent that it should. Processing can be accomplished with or without pressure and vacuum depending on the type and size of the specimen. All processing is done in the histology facility.
After the tissue has finished the infiltration step, it is removed from the cassette, and placed into a mold with liquid paraffin which is allowed to solidify. It is then ready to be sectioned and placed onto a slide.
The paraffin block is routinely sectioned at a thickness of 5 microns. The thickness of the section can be changed if requested.
Routine H&E (hematoxylin and eosin) staining is available at all times. Other special stains are done upon request. These include Trichrome for collagen, Bodians for nerve fibers, Luxol Fast Blue for myelin sheath, PAS for basement membrane and glycogen, Congo Red for amyloid, Oil Red O for fat and others that may be required for special projects.
Cutting frozen sections is sometimes best for doing immuno staining, especially when the antigen is masked by fixation. If frozen sections are required it is advisable to schedule an appointment.
Turnaround time for work submitted depends on the size of the project as well as the number of other projects that are being carried out at the time of submission. Often times the finished product can be obtained the next day and almost always within 3 days.
1. Place specimen in 5% sucrose solution - leave 4 hours in refrigerator.
2. Place specimen in 20% sucrose solution overnight in refrigerator.
3. Rinse specimen in OCT compound.
4. Place fresh OCT in mold and position specimen in mold.
5. Cover specimen with OCT.
6. Freeze specimen.
1. Immune a beaker containing enough isopentane (2 methyl butane-
Fisher #03551-4) to cover specimen in freezing mold in liquid nitrogen.
2. Place OCT in freezing mold in liquid nitrogen.
3. Cover specimen with OCT.
4. When isopentane is cold enough, a white precipitate will appear on
bottom of beaker.
5. At this time immerse entire specimen in isopentane using a forceps
to hold it straight.
6. When entire sample turns white it is frozen - do not allow it to remain in isopentane too long or it will crack.
7. Store sample at - 80 degrees.
1. If specimen has been fixed, rinse fixative off in running water or several changes of buffer.
2. Never use an alcohol-ice slurry to freeze. If alcohol contaminates the specimen, it will not freeze at -20. This will make cutting on the cryostat impossible.
3. Make sure specimen is oriented properly in OCT at the bottom of the freezing mold.
4. If there is more than 1 piece of tissue in the mold, be sure they are on the same level in the OCT.