THE CENTRAL MICROSCOPY FACILITY

Note: I have been told that it is possible to make the 3-aminopropyltriethoxysilane dilution into water! The half-life of the water-diluted "APTS" at pH 7, 24C is 8.4 hr.  This should be much more eco-friendly. This information was provided by Tom Phillips of the University of Missouri.  Check his website for the details......
http://www.biology.missouri.edu/faculty/phillips.html
http://www.biotech.missouri.edu/mcc/

AminoSilane-Glass Method

The following method is used to treat glass so that aldehydes, such as free aldehydes remaining after glutaraldehyde fixation, will be bound. This allows various materials to be immobilized for preparative treatments and microscopic examination. Exploration of this method came from a failure to achieve adequate retention of single-celled algal specimens using poly-L-lysine treated glass or various membrane filters. This method gave excellent results. A monolayer of firmly bound cells that were not easily displaced by vigorous agitation during SEM preparative steps was easily produced. Complete methods and references are included. I would like to thank those responding to a query to the Microscopy Society of America listserver for their suggestions, and Scott Whittaker for bringing this method back to my attention.

The basic method is simple. Clean, dry glass is treated with dilute (1-4% in dry acetone) 3-aminopropyltriethoxysilane for about 15 min, rinsed in dry acetone, then water. This produces covalently bound amino groups on the glass surface. Aldehydes will covalently bind to these amino groups. This treated glass can be used in two ways: by letting the free aldehydes of a glutaradehyde-fixed and rinsed sample react with the glass-amino groups, or by the indirect method of reacting glutaraldehyde with the glass-bound amino groups and then allowing the free aldehydes that remain to bind to amino groups of the sample proteins. This latter method could be used where glutaraldehyde fixation of the sample is not desired. I found an increase in cell binding as the concentration of aminosilane was increased from 1% to 4% (15 min treatment) and an increase in cell binding as the treatment time was increased from 5 to 15 min (2% aminosilane).

The simple method given above works. It is far less involved and time-consuming than the method of Ris and Malecki. In particular, the extended time and temperature of treatment they use clearly isn't required, but could be beneficial in some circumstances.

For samples being prepared for the SEM the glass can be heavily carbon coated and glow-discharge treated (to render hydrophilic) before the aminosilane treatment. The aminosilane appears to still bind well and the carbon results in excellent substrate conductivity for the applied samples that would otherwise depend on the sputtered film only to provide conductivity over the glass.

Tips

3-aminopropyltriethoxysilane is available from Sigma Chemical Co. and Aldrich Chemical Co. The chemical is stored at 4C and is sensitive to water so the stock bottle should be brought to room temperature before opening. Dispensing to small vials (Nalgene cryotubes are good) will avoid frequent opening the stock bottle.

The diluted (1%-4%) aminosilane in acetone is effective for at least a week if stored in refrigerator and warmed to room temperature before opening.

Small coverglass pieces can be floated on very small droplets (25 microliters or less!) of diluted aminosilane on a square of Parafilm (tm) in a Petri dish for the 15 min treatment time at room temperature. A bit of acetone in a vial cap will help saturate the atmosphere with acetone to reduce evaporation.

References

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