2/4/98

some weeks ago I asked for adresses for purchasing collodium. Thanks to all

who answered me!

Now I got pyroxilin (= collodium) 2% in amylacetate.

When I put one drop onto water, it spreads incredibly. So the resulting

film is much to thin and tears immediately when I get the grid into the EM.

Is the 2% solution to weak?

Is there another method to produce a thicker film?

I am trying the collodium support for I get enormous binding of the

*primary* antibodies to the formvar sometimes. I find it often, that

primary antibodies bind to formvar, but not to that extend that I saw with

two antibodies from eggyolk and rabbit. Any suggestions how to overcome

this?

Thanks a lot in advance

Birgit

Dr. Birgit Neubohn

Institute of Plant Genetics and Crop Plant Research (IPK)

Corrensstr. 3

D-06466 Gatersleben-Deutschland

Tel.: (+49) 039482 5447

Fax: (+49) 039482 5139

e-mail: neubohn@ipk-gatersleben.de

For doing immunoelectron microscopy I would suggest that if at all possible

you completely eliminate the collodion and formvar films.

Also, the kind of metal that is used in the grid seems to somehow affect non-

specific binding as well.

If it is a question of supporting your sections during the immunolabeling, I

would suggest you use a fairly high mesh (300 or 400 mesh) gold grid,

preferably with a hexagonal mesh pattern, with no supporting film. Just put

the sections on the grids, dry them, and begin the immunolabeling procedure.

If this is not possible, and you absolutely must use a supporting film, you

can try increasing the salt concentration in your buffer washing steps. If

you are using phosphate buffered saline or any other recipe with NaCl in it,

it is probably around 150 mM (0.9%). Try boosting the NaCl to 5X normal.

This would make it 750 mM, or 4.5% by weight.

If you use the high salt buffer, remember to incubate the grids in a couple of

changes of regular strength (150 mM) saline before you go to the next step, to

get the salt back into the range of physiologic strength! High Salt

concentrations will often get rid of non-specific binding.

I hope this is of some assistance. Let us know how it works.

Best regards,

Bob

*********************************

Robert (Bob) Chiovetti

E. Licht Company / USA / 1-800-865-4248

rchiovetti@aol.com

You didn't mention carbon coating your films after they were placed

on grids. This is necessary to dissipate the energy from the beam.

I try not to use films if it can be avoided. However, with

troublesome plant or bacterial samples it is often needed.

Hank Adams

Electron Microscopy Lab

New Mexico State University

Las Cruces,NM 88003

phone: 505-6463600

fax: 505-6465665

2% should be o.k., but collodine solutions have this wonderful

property in that you can stack the layers. I usually use 4%, I

gently place one drop on the watre surface, as you've noted as the

drop spreads you note that it reaches maximum spread and then

'bounces" back a little, right when it bounces back I add one more

drop to the middle. The second drop spreads on top of the first

giving a thicker film. You can use the color index (just like

sectioning) to tell you approximately how thick the film is and just

keep adding drops of the 2% until you get a color/thickness you like.

Richard E. Edelmann, Ph.D.

Electron Microscopy Facility Supervisor

352 Pearson Hall

Miami University, Oxford, OH 45056

Ph: 513.529.5712 Fax: 513.529.4243

E-mail: edelmare@muohio.edu

Try a smaller petri for spreading or two drops, interfernce

colors will help with judging thickness. Personally I make my own from the

parlodion sticks. Good luck.

Mike D

JHMI Microscopy Facility

delannoy@welchlink.welch.jhu.edu