APTS_Methods

THE CENTRAL MICROSCOPY FACILITY

APTS = 3-aminopropyltriethoxysilane AminoSilane-Sticky Glass Method

Note: Tom Phillips has pointed out that the water-sensitive decomposition is fairly slow so the APTS solution can be prepared in dH2O instead of "dry acetone" and this greatly simplifies the process.

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 initially learned about this method from Has Riis at a microscopy workshop at University of Wisconsin, Madison in 1996. 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, and Tom Phillips for the tip that the working solution can be effectivley prepared in water!

The basic method is simple. Clean, dry glass is treated with dilute (1-4%) 3-aminopropyltriethoxysilane for about 15 min, then rinsed in 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.

The diluted working solution can be prepared in water; the stock APTS material is water-sensitive and must be kept tightly closed and dry. Tom Phillips treats glass for only 1 minute in 2% aqueous dilution of APTS.

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 main 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. Diluted APTS in water should be used within a couple of hours.

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.

References

     J.P. Robinson, P. Dunhill, and M.D. Lilly, 1971. Biochim.Biophys.Acta 242, 659-661.
     M. Buechi and T. Baechi, 1979. J. Cell Biol. 83, 338-347.
     Malecki and Ris, 1990. Scanning 13, 82.
     Malecki and Ris, 1992. Scanning 14, 76 LI>Malecki, 1991. Scanning Microscopy, Supplement 5, S 53
     Ris, 1991. EMSA Bull.21, 54
     Ris and Malecki, 1993. J. Struct. Biol. 111, 148.

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*Tom Phillips APTS method.

Thomas E. Phillips wrote:

Yes, I am familiar with the older method requiring dry acetone. But the half life of APTS in water pH 7 at 24 C is 8.4 hrs (see nice summary of chemical properties and safety at http://www.inchem.org/documents/sids/sids/919302.pdf ). So my advice is make it fresh and skip the hassles with acetone. This means you can use plastic slide holders to dip!

Here is my protocol (written in excruciating detail for the freshman students who do this for me!):

Supplies:
2 x 2000 ml Glass Beakers
1 x 250 Schott Bottle (labeled APTS Container)
1 x 250 ml Graduated Cylinder
1 x 100~1000 microliter pipette
APTS (3-aminopropyltriethoxysilane – Acros #151081000) solution TOXIC!!!
White plastic dipping jar
Slides in holders
Timer

Procedure: (All procedures done in the fume hood)

   1. Fill the 13 plastic slide holders (plastic ones from Electron Microscopy Sciences) with clean microscope slides.
   2. Fill two 2000 ml glass beakers each to ~1600 ml with deionized water.
   3. Place beakers to the side.
   4. Fill 250 ml graduated cylinder with 125 ml of deionized water and pour into 250 ml Schott bottle labeled APTS container.
   5. APTS solution is stored in the left side refrigerator – top shelf. Transfer it to the fume hood. Wear gloves for these steps. We want a 2% APTS stock solution which requires us to add 2.5 mls of APTS stock to the 125 mls of dH20 in the APTS Schott bottle. It is most convenient to do this with 3 aliquots of 833 l of APTS stock using a micropipette. Gently mix the water and APTS by capping and gently swirling the solution in the Schott bottle. Return APTS bottle to refrigerator as soon as you are done using it.
   6. Fill the white holding container to black line with 2% APTS solution. Keep the 2% APTS solution close to refill the white holder when the level drops.
   7. Place the white holding container and two 2000 ml glass beakers w/ water in a row.
   8. Start the timer and place a slide holder with slides in the white container with 2% APTS solution. Allow to sit for 1 min. We generally set a timer for 35 min and proceed to the next step every minute.
   9. After 1 min, remove the slides from the white container and begin swirling them in the first of the 2000 ml beakers of water. (You do not need to submerge completely. Keep the colored tops of the slides out of the water while swirling.) Do this for 1 min.
10. After rinsing for 1 min, repeat this step in the second 2000 ml beaker of water for an additional minute.
11. After the two rinse steps, place the slides in a clean stop to air dry.
12. Repeat steps 13-16 for all slides. Remember to keep the APTS solution in the first chamber at the level of the black line.
13. Once the slides have air dried, transfer to a slide box and heat overnight in the oven. 100 C is probably best but I must admit I often use 60 C since the plastic slide boxes probably can’t tolerate higher than 60ºC.

Thomas E. Phillips, PhD
Professor of Biological Sciences
Director, Molecular Cytology Core
2 Tucker Hall
University of Missouri
Columbia, MO 65211-7400

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