The silane compound comes
as a liquid from Sigma. Its name is:3-aminopropyltriethoxysilane
slides in a 2% silane/acetone solution for one minute
slides in 100% acetone for 1 min.
slides in ddH2O for 1 min.
this step with agitation
- Air dry.
The slides seem
consistently sticky in our hands, although we once had trouble with really
teeny weenie sections that someone here was cutting (but this was also for an
application with lots of long rinses in running water). Further information
about this can be found in:
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.
--Tobias I. Baskin
University of Missouri, Biological Sciences, Tucker Hall Columbia, MO 65211 USA
We would like to tell you
about a method that we have now used routinely for about 5 years. It is based
on procedures published by :
JP Robinson, P. Dunnill,
and MD Lilly, in Biochim. Biophys. Acta 242, 659-661, 1971, and M. Buechi and
T. Baechi, in J. Cell Biol. 83, 338-347, 1979.
- The glass
(or other carriers) must be absolutely clean. This can be accomplished by
immersing for several hours in aqueous 20% sulfuric acid, or 20%
hydrochloric acid in ethanol. I prefer washing with Bon Ami cleaning
powder until the surface is hydrophilic. The clean carriers are soaked for
2 h in anhydrous acetone that had been stored for at least 24 h over
- Prepare a
2% solution of 3-aminopropyltriethoxysilane (Aldrich Chemical Co
Milwaukee WI) in anhydrous acetone.
carriers are placed in appropriate containers (glass, Nalgene,
polypropylene) which must be totally filled with the silane solution , and
stored for 24 h at 50 degree C. If you use a 4% solution of silane, the
carriers will be ready for use in 12 h. The silane treatment coats the
carriers with amino groups.
- To attach
fresh cells or cell fractions to the carriers, they are rinsed in acetone
and immersed for one hour in 1% glutaraldehyde. The carriers are then
placed into distilled water at 4 degree C. They can be kept for about 5
days. Their surface is now coated with aldehyde groups, which binds
covalently with aminogroups on the surface of cells or cell fractions. If
cells or cell fractions must be fixed before attachment (in glutaraldehyde
fixative) the 1% glutaraldehyde is omitted. Exposed aldehyde groups on the
fixed biological materials will bond to the amino groups on the carriers.
We have used sticky
carriers prepared by this procedure to attach cells previously fixed in
suspension to glass carriers:
imaging by field emission SEM (Malecki and Ris 1990, Scanning 13 ,82 ; Malecki
and Ris 1992, Scanning 14, 76; Malecki,1991, Scanning Microscopy,Suppl.5,
treated coverslips were used to attach isolated amphibian oocyte nuclear
envelopes for imaging of nuclear pore complexes by FESEM (Ris, EMSA
Bull.21, 54, 1991. )
- Such coverslips
were also used to retain sections of epon embedded biological materials,
after extraction of the epon for FESEM imaging (Ris and Malecki, 1993. J. Struct.
--H. Ris and M. Malecki,
Integrated Microscopy Resource, Univ. of Wisconsin, Madison WI. firstname.lastname@example.org - Malecki@macc.wisc.edu