Tissue Processing and Sectioning
Decalcification of bone:
After fixation bone, must be
decalcified or else it
won't cut on the microtome:
- Immerse tissue cassette in 11% formic acid with a stir bar overnight in a fume hood.
- Rinse in running water for 30- 60 minutes (the smell should be gone).
Storage in 70% Ethanol
After adequate fixation tissues are
transferred to
70% ethanol and may be stored at 4°C.
Paraffin processing
The automated 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:
- 70% Methanol 1 hour (x1)
- 90% Methanol 1 hour (x1)
- 95% Methanol 1 hour (x1)
- 100% Methanol 1 hour (x1)
- 100% Ethanol 1 hour (x1)
- Xylene 1 hour (x2)
- Paraffin (65°C) 30 min. (x1)
- Paraffin (65°C) 30 min (x1) with vacuum (applied manually on our machine).
If the processor is to be run overnight it should be programmed to hold on the first EtOH 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
tissues 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.
Sectioning tissues
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.
