[3dem] Response to 3dem Digest, Vol 62, Issue 3 message 2
Robert Marshall
rmarshal at bluebottle.com
Fri Oct 12 12:34:09 PDT 2012
I've attained super success over Formvar using
PEI instead. A precise and detailed description
is found at
http://em3d.org/EM3D/downloads/PEI.pdf. These
films are stronger than Formvar and PEI films can
be stripped off the slides many weeks after
dipping, unlike Formvar.
Robert Marshall
Research Associate
Biology
Texas A&M Univ.
>
>
>------------------------------
>
>Message: 2
>Date: Thu, 4 Oct 2012 15:57:10 +0000
>From: "Sharon G. Wolf" <Sharon.Wolf at weizmann.ac.il>
>Subject: RE: [3dem] prepare Formvar Grids
>To: Denis Chaix <denis.chaix at ibs.fr>
>Cc: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
>Message-ID: <3A7444B2D486E2419D240120CA143DC4597F4AC1 at IBWMBX02>
>Content-Type: text/plain; charset="iso-8859-1"
>
>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.
>Best,
>Sharon
>-----------------------------
>Sharon Grayer Wolf, PhD.
>Electron Microscopy Center
>Weizmann Institute of Science
>Rehovot 76100 Israel
>tel: 972-8-934-4421
>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
>
>Hi Denis,
>
>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.
>Cheers,
>
>Patricia
>
>
>
>
>
>On Sep 28, 2012, at 1:36 AM, Peter Engelhardt
><Peter.Engelhardt at Helsinki.Fi<mailto:Peter.Engelhardt at Helsinki.Fi>>
>wrote:
>
>
>Hi Dennis,
>
>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
>
>Cheers
>Peter
>
>
>On Sep 27, 2012, at 11:30 PM, Denis Chaix wrote:
>
>
>Hello
>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
>45ƒ 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.
>
>Thanks
>
>
>
>Denis
>--
>--
>____________________________________________________________________________
>_______________
>
>Chaix Denis <denis.chaix at ibs.fr<mailto:denis.chaix at ibs.fr>>
>Microscopie Electronique et MÈthodes
><http://www.ibs.fr/groupes/groupe-microscopie-electronique-et/>
>Room 6234
>Institut de Biologie Structurale UMR-5075 <http://www.ibs.fr/spip.php>
>41 Rue J. Horowitz
>38027 Grenoble Cedex 1,France
>Tel: +33.4.38.78.01.59.
>Fax: +33.4.38.78.54.94.
>____________________________________________________________________________
>_______________
>
>_______________________________________________
>3dem mailing list
>3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>
>https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
>
>_________________________________________________________
>Peter Engelhardt, PhD, docent
>Adjunct Professor in Molecular Genetics
>Unit of Molecular Electron Tomography
>
>Department of Pathology
>Haartman Institute
>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
>Finland
>
>Home address:
>Lindstedstv”gen 1 B 7
>FI-02700 Grankulla,
>Finland
>
>Mobile: +358-(0)400193342
>Email: Peter.Engelhardt at Helsinki.Fi<mailto:Peter.Engelhardt at Helsinki.Fi>
>URL: http://www.lce.hut.fi/~engelhar/
>
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>_______________________________
>Patricia Grob, Ph.D.
>Research Specialist
>Nogales Lab
>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
>URL: cryoem.berkeley.edu<http://cryoem.berkeley.edu/>
>Email: pgrob at berkeley.edu<mailto:pgrob at berkeley.edu>
>
>
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