[3dem] prepare Formvar Grids
Sharon G. Wolf
Sharon.Wolf at weizmann.ac.il
Thu Oct 4 08:57:10 PDT 2012
By coincidence, I had to prepare for the first time in many years some Formvar grids. I totally failed using ANY of the "fool-proof" recipes discussed in this thread. I spoke to the folks on staff here at the EM Unit who have been doing it routinely for many years, and they talk about the need for very "dirty" glass slides (they sequester "special" boxes in secret places), of how one has to score the glass a certain way with a razor blade, etc etc, and even THEN, there is a high failure rate. My colleague Eyal Shimoni mentioned that he had tried using hand soap on the glass, with some success. After integrating this information, I came to the conclusion that, indeed, it is clear that surfactants are the key to getting the Formvar off. So perhaps providing the surfactant, as Eyal suggests, is a good idea for getting reproducible results, and removing the black magic and frustration from this procedure.
I tried the following protocol on three different types of glass slides, and IT ALWAYS WORKED. The answer does lie with the surfactant:
(1) Take a pre-cleaned slide straight out of the box, and smear a little dab of liquid hand soap on each side.
(2) Use lens paper to wipe off the glass slide. I really rubbed off all visible traces of the soap off, leaving no streaks behind.
(3) I dipped the slides in 0.25% Formvar in ethylene dichloride, and blotted off the excess on the edges with kimwipes
(4) I scored the glass slide on the sides and bottom with a razor blade
(5) The formvar EASILY slid off onto the water surface
(6) I checked these grids in the electron microscope. Nice and clean and intact.
Hope this can help others, too.
Sharon Grayer Wolf, PhD.
Electron Microscopy Center
Weizmann Institute of Science
Rehovot 76100 Israel
sharon.wolf at weizmann.ac.il
From: 3dem-bounces at ncmir.ucsd.edu [mailto:3dem-bounces at ncmir.ucsd.edu] On Behalf Of Patricia Grob
Sent: Friday, September 28, 2012 8:27 PM
To: Denis Chaix
Cc: 3dem at ncmir.ucsd.edu; Peter Engelhardt
Subject: Re: [3dem] prepare Formvar Grids
We were having the same problem getting Formvar to come off so called "pre-cleaned" glass slides for a while. We tried a lot of black magic in desperation (one protocol included "pizza breath" or "garlic breath") until we found one technique that has been 100% foolproof in our hands:
Before dipping glass slides into Formvar solution (for us it works both for 0.5% Formvar in chloroform or 0.5% Formvar/chloroform with 50% glycerol droplets in suspension - used for holey films):
- Dip entire glass slide into pure ethanol or methanol beaker for a few seconds (it may also work with other solvents but those are the only ones we have used so far)
- take slide out, wipe off excess solvent with Kimwipes.
- Repeat last two steps if you still see streaks on glass
- blow lint off with compressed air if needed
- let slide finish drying for 2-5 min
Make sure to float the Formar off within 1/2 hr of cleaning!
we then proceed as follows:
- dip clean glass slide into Formvar solution, blot edges on filter paper, let dry (a few minutes are sufficient). Again, use within 1/2 hr!
- score edges with new razor blade
- enter slide at 45 degree into clean (Millipore) water to float Formvar film off glass.
I hope this will work for you as well.
On Sep 28, 2012, at 1:36 AM, Peter Engelhardt <Peter.Engelhardt at Helsinki.Fi<mailto:Peter.Engelhardt at Helsinki.Fi>> wrote:
We use simple almost "magic" foolproof method - originally shown by Jan Derksen visiting our lab in1970ties.
- Polish slides with a clean cotton towel extensively that is all corners etc
- Ready when you experience that the slide appears more slippery after polishing - might take some time.
- Blow away the lint that might appear.
- Proceed instantly accordingly to any of the protocols presented.
The procedure helps when the air is very dry - e.g.in winter time
and in labs for cryo-EM works where the humidity is kept purposely low.
Let's hope this trick helps
On Sep 27, 2012, at 11:30 PM, Denis Chaix wrote:
We have actualy some troubles to prepare Formvar films.
In fact it's impossible to release the film from the glass slide.
Here you can find our protocol
Formvar solution. 0,4% in chloroform .
Pour the solution into a clean Coplin jar
Dip pre-cleaned slides ( rub thoroughly with Kimwipes) into beaker.
Drain corner on side of beaker.
Blot edge of slide on filter paper.
Score the edges of the Formvar film with an acetone-cleaned razor blade.
Breathe over the length of the slide to loosen the film.
Float the film off onto a clean water surface by slowly immersing the slide into the water at a 45° angle.
Have you some tricks or tips to improve this protocol and help the release of the film ?
For example, have you some ideas like special brand of slides, or a way to clean the slides.
Chaix Denis <denis.chaix at ibs.fr<mailto:denis.chaix at ibs.fr>>
Microscopie Electronique et Méthodes
Institut de Biologie Structurale UMR-5075 <http://www.ibs.fr/spip.php>
41 Rue J. Horowitz
38027 Grenoble Cedex 1,France
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Peter Engelhardt, PhD, docent
Adjunct Professor in Molecular Genetics
Unit of Molecular Electron Tomography
Department of Pathology
P.O.Box 21, FI-00014 Univ. of Helsinki, Finland
Visiting Address: Haartmaninkatu 3, Helsinki
Nanomicroscopy Center (NMC ), Department of Applied Physics
Aalto University, School of Science and Technology,
Puumiehenkuja 2, FI02150 Espoo
Lindstedstvägen 1 B 7
Email: Peter.Engelhardt at Helsinki.Fi<mailto:Peter.Engelhardt at Helsinki.Fi>
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Patricia Grob, Ph.D.
HHMI at UC Berkeley
Molecular & Cell Biology Department / QB3,
Stanley Hall #742
University of California, Berkeley
Berkeley, CA 94720-3220
Phone: (510)666-3335 Fax: (510)666-3336
E.M. suite B307: (510)666-3339
Email: pgrob at berkeley.edu<mailto:pgrob at berkeley.edu>
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