6/10/97

Hello all,

What is the best way to process cells for TEM which are grown on

glass

coverslips affixed to plastic petri dishes. I will need to remove

the

glass from the plastic and cut ultra thin sections. I have

previously

tried to do this and could not remove the glass. I gave up on

glass and

started using aclar. I now must return to using glass. Any

suggestions?



Sally

Sally Shrom

sally@retina.anatomy.upenn.edu

Sally:

Our EM facility has been using glass coverslips in several tissue

culture

experiments. After flat embedding with epoxy/alradite resin, we

remove the

glass, using cold HF[hydroflouric acid] (in a Fume Hood!) for

@4-8 minutes. We

get 100% success rate, using many different types of glass

coverslips. However,

the glass cover slips are not attached to plastic petri dishes.

Do the

coverslips need to be attached to petri dishes? The glass surface

has to be

exposed to the HF fluid. You can still use this technigue if your

coverslips

are atteched. you will have to add an extra step to remove the

glass from the

plastic, via sandpaper. Please contact me, if you would like a

detailed step by

step procedure for this technique. It is very straightforward.



The references we started with:

Moore, M.J., 1975, Removal of Glass Coverslips from Cultures Flat

Embedded in

Epoxy Resins Using HF, . Microscopy, v. 104, p. 205.



Rieder, Conly and Bowser, Samuel, 1987, "Correlative LM and EM on

the Same

Epoxy Section", in Correlative Microscopy in Biology ,

Chapter 11, Academic

Press, 1987.



-Louisa howard

Dartmouth College

EM Facility

Hanover, NH. 03755

(603) 646-3492

Louisa.Howard@ Dartmouth.edu

Separating glass from plastic is easy in my experience after

thermal shock.

Immerse the specimen in liquid nitrogen for a few seconds.

Jim Darley



ProSciTech Microscopy PLUS

PO Box 111, Thuringowa QLD 4817 Australia

Phone +61 77 740 370 Fax: +61 77 892 313

Great microscopy catalogue, 350+ Links, MSDS

************************ http://www.proscitech.com.au

Have you tried using Thermanox coverslips? Not cheap, but they

are good for

culturing and good for embedding and they peel away from resin

with the

application of a little heat from a hot-plate. Thermanox can also

be

sectioned along with the resin if you like, but I have had

problems with

this because the Thermanox and resin do not stretch to the same

degree, so

you end up with sections wrinkled on one edge.



Regards



Stephen Griffiths

s.griffiths@ucl.ac.uk

Visual Science Department Phone:- 0171 608 6914

Institute of Ophthalmology Fax:- 0171 608 6850

Bath Street, London. EC1V 9EL

Jim,

I tried immersing my plastic in liquid nitrogen and was unable to

remove

the glass. Perhaps you did something special during the whole

process of

embedding, or perhaps I am not using the best plastic. I use

embed 812

kit. Also, I have heard that if you use serum in the culture

medium, then

the glass will not come off the surface of the plastic. Does this

make

sence?

Sally Shrom

sally@retina.anatomy.upenn.edu

We have "popped" cells grown in both serum and serum-free medi on

glass

coverslips using liquid nitrogen 100's of times. serum makes no

difference. The coverslips are fixed either in the 12 well

plastic trays

or transfered to 20 ml glass scint vials. by the time we get to

the

dehydration steps, we always switch to the glass vials. after

100%

ethanol, we infiltrate with embed 812 (1:1 with ethanol

overnight) then

100% resin for 3 hrs then place the coverslip cell side up on a

glass slide

with the thin layer of resin that comes with it when we transfer

to the

slide (you don't want it too thin or too thick but it if you are

in doubt,

simply add 1 drop to the coverslip after transfering and let it

flow

naturally. you shouldn't get a final plastic spread of more than

1.5 - 2

times the diameter of the coverslip or you are using too much.

when you

take the slides out of the oven, let them cool for 15 min and

touch with a

razor blade. if you get a gooey strand when you pull the blade

away, you

haven't polymerized it enough. if it is not too gooey,

cross-hatch the

surface of the plastic using the razor blade to score deeply into

the

plastic (to the level of the glass). SLOWLY immerse the slide

into Liquid

N2 and the squares should pop right off. look at the surface of

the

coverslip and bottom of the square to ensure no glass is going

with the

square. if so, you have over polymerized. for some projects, we

simply

put the square in the flat microtome holder and cut it but

ususally we

re-embed the square in a standard mold. if you aren't

re-embedding, you

should heat the squares in a rubber mold overnight. once you

have the

timing of how long to polymerize the coverslips, it is trivial.

we havent

had a failure in years of doing this a lot of times. good luck.



Tom Phillips



>Jim,

>I tried immersing my plastic in liquid nitrogen and was unable

to remove

>the glass. Perhaps you did something special during the whole

process of

>embedding, or perhaps I am not using the best plastic. I use

embed 812

>kit. Also, I have heard that if you use serum in the culture

medium, then

>the glass will not come off the surface of the plastic. Does

this make

>sence?

>

>Sally





Thomas E. Phillips, Ph.D.

Associate Professor of Biological Sciences

Director, Molecular Cytology Core Facility

3 Tucker Hall

University of Missouri

Columbia, MO 65211

(573)-882-4712 (voice)

(573)-882-0123 (fax)

[Return to Tips & Tricks Menu]