M. Fero 12.23.2011
This protocol replaces our older methanol based procedure
The usual fixative for paraffin embedded tissues is neutral buffered formalin (NBF). 10% NBF contains 4% formaldehyde by weight and is therefore equivalent to 4% paraformaldehyde. NBF contains a pH buffer plus a preservative (methanol) which prevents the conversion of formaldehyde to formic acid. Because of the preservative, NBF has a shelf life of months, whereas 4% PF must be made fresh.
Optimal formalin fixation requires adequate time to form -CH2- crosslinks, generally ≥ 48 hrs, at room temperature, for 2 mm-thin sliced tissues. Inadequately fixed tissues will become dehydrated during tissue processing, resulting in hard and brittle specimens. Alcohol based fixatives generaly do not give good morphology but may be useful for special cases (such as BrdU staining). A particular challenge is immunostaining fixed specimens. In many cases formaldehyde fixation will prevent recognition of epitopes by the primary antibody. Occasionally, "antigen retrieval" procedures will improve results. Usually frozen sections are a better bet for immunostaining, but at the expense of morphology. An alternative approach, suitable for thin or porous tissues, is to perform immunohistochemistry on fresh tissues and then post-fix and embed the tissues in paraffin.
B5 is a formalin fixative that also contains 6% (w/v) mercuric chloride. It is gives superior morphology for lymphoid tissues, but care must be taken not to over-fix tissues (fixation time = 2-12 hours). Becuase it contains mercury, B5 should not be used routinely. Care must be taken to minimize volumes and collect all used solutions for hazardous waste disposal.
Decalcification (only necessary for samples containing bone):
After fixation, bone must be decalcified or else it won't cut on the microtome.
Storage in 70% Ethanol
After adequate fixation, tissues may be transferred to 70% ethanol and stored at 4°C for 1 week. This should only be done for a short time, prior to paraffin processing. The main goal is to avoid contamination of processor solutions with fixative.
The Shandon Hypercenter XP tissue processor takes the cassettes through a series of graded EtOH baths to dehydrate the tissues and then into xylene. Hot paraffin can then permeate the tissues:
If the processor is to be run overnight it should be programmed to hold on the first ethanol bath and not finish until the next morning so the specimens do not sit in hot paraffin longer than the time indicated. If specimens are fresh they may incubate in formalin in the first stage on the machine. It is important to not keep the tissues in hot paraffin too long or else they become hard and brittle. The vacuum helps to speed up the permeation of tissues by paraffin and helps get rid of any small air bubbles. Processed tissues can be stored in the cassettes at room temperature indefinitely.
Embedding tissues in paraffin blocks
Tissues processed into paraffin are melted by placing the entire cassette in 65°C paraffin bath for 15 minutes. Turn the heat block on to melt the paraffin one hour before adding the tissue cassettes. Also warm metal block molds on the hot plate. The paraffinzed specimens may be dissected with a razor blade and placed with the cut surface down towards the bottom of the mold. Hot paraffin is added to the mold and from the paraffin pot. Use heated forceps to orient the tissues in the mold. When the tissue is in the desired orientation add the labeled tissue cassette on top of the mold as a backing. Be sure there is enough paraffin to cover the face of the plastic cassette. Slide the mold off of the hot plate onto a cold aluminum heat sink. When the wax is completely cooled and hardened (~20 min.) the paraffin block can be popped out of the mold. If the wax cracks or the tissues are not aligned well, simply melt them again and start over. Tissue blocks can be stored at room temperature for years.
Turn on the water bath and check that the temp is 35-37ºC. Use fresh deionized water (DEPC treated water must be used if in situ hybridization will be performed on the sections). Blocks to be sectioned are placed face down on an ice block or heat sink for 10 minutes. Place a fresh blade on the microtome. Insert the block into the microtome chuck so the wax block faces the blade and is aligned in the vertical plane. Set the dial to cut 4-10 µM sections. The blade should angled 4-6º. Face the block by cutting it down to the desired tissue plane and discard the paraffin ribbon. If the block is ribboning well then cut another four sections and pick them up with forceps or a fine paint brush and float them on the surface of the 37ºC water bath. Float the sections onto the surface of clean glass slides. If the block is not ribboning well then place it back on the ice block to cool off firm up the wax. If the specimens fragment when placed on the water bath then it may be too hot.
Place the slides with paraffin sections in a 65°C oven for 20 minutes (so the wax just starts to melt) to bond the tissue to the glass. Slides can be stored overnight at room temperature.