Sectioning and Mounting Protocol
Technovit Glycol Methacrylate: Technical Information and Help From
Heraeus Kulzer.
Sectioning
Sectioning is best done with a rotary or sledge microtome
such as the JB-4 Microtome. The features to look for are
retraction of the specimen on the return stroke and motorized motion
of the sample relative to the knife edge. Retraction is needed to
keep the hard sample from brushing against the knife edge on the
return stroke, damaging the edge and block face. Motorized sample
movement is desirable to get more reproducible cutting speed and
force.
The best sections are obtained on these microtomes
with a glass or tungsten carbide knife. Disposable
tungsten carbide knives are available from Energy Beam Sciences
which offer the ability to cut routine serial 1 micron thick sections.
Dry block faces are used; in fact, moisture and humidity will soften
GMA blocks sufficiently to make sectioning impossible. If the block
is too soft to section dry the block in a warm oven or move to a
less humid work environment.
Sections are collected from the knife edge using
tweezers and stretched by floating on a water bath. Two factors
are important when preparing
the water bath: cleanliness and temperature. Any residue of soap
or oil will decrease the surface tension of the water dramatically
reducing its stretching ability and/or causing the section to sink.
Increasing temperature also reduces the stretching ability of the
bath so that at 20° C stretching of 10 - 13% is possible while at
60° C stretching of 7 - 9% is possible. Section stretching allows
recovery of almost all the compression caused during sectioning.
Properly stretched, much less than 1% of the compression in vertical
dimension remains in the sections.
Most procedures call for mounting your sections on slides before
staining. The exception may be when staining sawn sections since
these retain enough strength to be held in tweezers and hand-dipped
in the various staining solutions (followed directly by coverslipping
to slides). Mounting thinner sections to slides before staining also
helps to prevent folds from developing in the sections. If you are
using slides precoated with silane, skip the
protocol for coating your own.
Solutions:
The silane material comes in a liquid form from Sigma. The full
name is 3,aminopropyltriethoxysilane.
Procedure:
- Dip slides in 2% silane/acetone solution for one minute.
- Dip slides in 100% acetone for one minute.
- Dip slides in double distilled water for one minute.
- Repeat step 3 with agitation.
- Air dry.
- Apply sections on a drop of water on the slide.
- Dry on 60°C hot plate for 2 - 5 minutes.
- Air dry at room temperature overnight.
These silanized slides can be stored at room temperature for months
without any problems.
Source:
Tobias Baskin, Univ. of Missouri, 109 Tucker Hall, Columbia, MO,
Email: baskin@biosci.mbp.missouri.edu
Citation:
Angerer, L. M. & Angerer, R. C. (1991) Localization of mRNAs
by in-situ hybridization. Functional Organization of the Nucleus:
a Laboratory Guide. Methods in Cell Biology, Vol 35, (ed. by B. A.
Hamalko & S. C. R. Elgin), pp. 37-71. Academic Press, San Diego.
Dip your coated slide into the waterbath under
the section and lift the section off the water surface. Dry the
slides for about 15 minutes
on a 60°C slide warmer. |
