1-7-99

I need assistance with flat embedding of rat cerebellum

(IEM-DAB), that is 100 um thick and 1.5 x 0.5 cm dimensions. Basically

we are worried about tissue curling during the dehydration. My first

inclination is argarose embed (before epon), but I would take any expert

advise.

MICHAEL DELANNOY

delannoy@welchlink.welch.jhu.edu

I embed vibratome sections as slides and then remove areas of interest for

re-embedding.

I use a Teflon Release Spray for coating the slides. It is available from

RS Components in the UK. It works very well. There may be an equivalent

available in the USA.

You just spray it on the slides, polish gently with a cloth and that is

all. No baking needed.

You could also try using Silicon coating using Sigmacoat. The solvent is

not nice but used with care it works very well for releasing coverslips

from "embedded" sections on slides.

I just dip the coverslips in the liquid allow to air dry and use them

straight away.

The coverslips usually lift off the section very easily and the section or

any part of it is very easily released from the slide.

Stephen Griffiths

Visual Science Department

Institute of Ophthalmology

Bath Street, London. EC1V 9EL

e-mail:- s.griffiths@ucl.ac.uk (work address)

or stephen.griffiths@dial.pipex.com (home address)

put them into flat molds standing up on their edges, but they would fall

over sometimes. We now cut the pointed end off a BEEM capsule; snap the

cap on the other end and wrap the junction with a sliver of Parafilm to

prevent leaking; put a drop of

resin in the lid (now upside down, with the Beem capsule sticking

upwards); and lift the thick section into the lid with a wooden

applicator stick broken into a flat wedged end with the tip then bent up

into the shape of a hoe then fill the capsule with resin. Do this on a

light box and under a dissecting scope.

If you need to keep straight which side is which, you can trim the tiny

thick section with a razor blade into a funny shape that you will

recognize. We use a trapezoid-like shape/state of Nevada shape:

That way you can keep the side of interest outward, e.g., confocal

scope-selected areas: Miller SE, Levenson RM, Aldridge C, Hester S, Kenan

DJ, Howell DN. 1997. Identification of focal viral infections by

confocal microscopy for subsequent ultrastructural analysis. Ultrastruc.

Pathol. 21:183-193.

Your sections are wider; thus, you will have to embed in a larger mouthed

container if you want sections parallel with the face. But the principle

is the same. If the sections have enough room to "swim" in the resin,

you can gently stretch them out with the applicator stick, i.e., they won't

premanently curl even though they will be flimsy as they float around in the

resin.

Sara E. Miller, Ph. D.

P. O. Box 3020

Duke University Medical Center

Durham, NC 27710

Ph: 919 684-3452

FAX: 919 684-8735

saram@duke.edu

we routinely embed flat sections

after the immuno proceedures, do any post fixation (OsO4), followed by

dehydration, infiltration with resin, then to embed we place the 100

micron section between two glass slides * one of which has been subbed

with a gelatin material, the other whioch has been subbed with Trenmittel

(we purchase ours from EMS) the trenmittel is something like teflon, it is

a release agent. remove all exess resin from around the section, then

clamp the two slides together (clothes pins or large paper clips)

polymerize as usual, then using a knife blade pry the two glass slides

apart, this takes a little practice.

finally reembed by filling a gelatin capsule with resin, let it thicken a

little, then invert it over thetissue section which remained on the subbed

slide, polymerize overnight. to free the section/block; hold slide over an

alcohol burner flame (use pliers) then after 6-8 seconds snap off the

block with the aid of haemostats or pliers. it really can work quite well.

but practice first it is a little tricky

MIKE ROCK

merock@du.edu

You shouldn't have much trouble with curling during fixation and

dehydration. The problem comes with getting absolutely flat sections for

polymerization. Your sections are a bit thicker than I used and we were

usually able to cut down the area of interest further but try this procedure. I

think it should work fine. I have used it to embed vibratomed x-sections

of rat brain which had been subjected to pre-embedding ICC reactions. In

this case, since the amount of reaction diminished as you cut further into

the section, it was critical to have absolutely flat material for serial

sectioning.

Try embedding in droplets of resin on a 22x22mm plastic coverslip

(available through most of the EM supply companies). Cover with another

coverslip and weight with metal washers or nuts. The weighting is important as

it really pressed the sections down insuring that they are very flat and

squeezes out extra resin. After 24 hr. polymerization, cut edges of cover

slips and they can then easily be separated. Cut the end off of gelatin

or beam capsules to give a tube. Place the capsules over areas of

interest on the sections, add one small drop of resin and polymerize a number of

hours to secure capsules to the sections. Then fill up the capsules and

finish polymerization.

When ready to section, blocks are easily broken or cut off of the

coverslips. There will be very little extra resin to cut through before

getting into the tissue making sectioning fairly easy once you are properly

lined up.

An alternative method is to embed between teflon-coated glass slides

which are then also weighted. I have found this a bit more cumbersome

than using the plastic coverslips but it may work better for thicker specimen

material.

Debby Sherman, Manager Phone: 765-494-6666

Microscopy Center in Agriculture FAX: 765-494-5896

Dept. of Botany & Plant Pathology E-mail: sherman@btny.purdue.edu

Purdue University

1057 Whistler Building

West Lafayette, IN 47907-1057

between two pieces of Aclar. If a minimum of resin is placed between

the sheets, the sections won't move much as a weight is applied to

flatten the sections. There are different grades of Aclar, and I had to

go directly to Allied Signal to get the one I prefer. The Aclar peels

easily away from the polymerized resin, leaving one with a "section"

that can be viewed with a LM for subsequent trimming. I super glue it

to a blank beem capsule for thin sectioning.

Best of luck,

Randy

--

Randy Nessler rnessler@emiris.iaf.uiowa.edu

Views expressed are my own.

http:\\www.uiowa.edu\~cemrf

To avoid tissue curling try 50% or 30% ETOH in the first dehydrating step.

You can do dyhradation in cell-culture multiwells.

You ll have to remove the specimens if you d like to use Propylene oxide

because this interacts with the multi-well plastic.

If you like to embed the sections flat, do it on silicated glass slides(or

try a slide, coverd with slightly fatted aluminium foil) , put a drop of

Epon/araldite on it, leave it at 45 C in the oven (12-24h) and then put BEEM

Capsules filled with Epon/araldit on the top(take care of bubbles!) ,

polymerize 2 days at 60 C. If you can t remove the glass slide easily, try

it with fluid nitrogen.

Good luck,

Michael Reiner

a2811111@smail.Uni-Koeln.DE