8/26/97

I have to embed some Drosophila melanogaster heads for transmission

electron microscopy and prepare the same for scanning electron

microscopy. The person requesting the work is interested in eye

morphology. I have done a literature search and have come up with some

relevant papers but my problem is that I will probably get these heads

before I get the papers!! Working in the medical field, I have never

embedded a sample with an exoskeleton before and am wondering if someone

out there can give me an idea on the appropriate fixatives to use

including fixation times and any little tricks I may need to know. Do

the heads sink in the fixative or do I need to spin them down or use a

vacuum? Is one resin better than another? How do they cut? Also, for

SEM, which fixative do I use and for what duration? Again, are there

any tricks to the dehydration and critical point drying procedures?

Thanks in advance

Sarah Ellis

sarahe@raid.res.petermac.unimelb.edu.au

I spend my time embedding triatomino's legs. This is my method:

1)cut the heads

2)put 2-3 hs. in fixative medium ( I don't use transmition mic.)

3)deshidratation:

ROH 70% (1*10 min)

-- 80% ----

-- 90% ----

EtOH 100%(3*10 min)

4)EtOH 100% + Propilenoxide : (1:1) : ( 10-15 min)

5)Propilenoxide (10-15 min)

6)Propilenoxide + Durcupan (epoxi resine): (1:1) : ( 1 night)

8)open the recip. at 40 centigrade degrees ( 1h. 30 min)

9)put in new Durcupan at room temperature (1h.)

10)Put in new durcupan and orientation the head (1 night at 60 cent. degrees)

Veronica Campanucci

--------------------------------------------------------------------------------

Veronica Andrea Campanucci

Laboratorio de Fisiologia de Insectos

Dpto. de Ciencias Biologicas

Facultad de Ciencias Exactas y Naturales Tel (54 1) 781-5021 to 29, ext. 332

Universidad de Buenos Aires FAX (54 1) 782-0582/544-7893

(1428) Buenos Aires e-mail: veronica@bg.fcen.uba.ar

Argentina HTTP://biolo.bg.fcen.uba.ar/physinse.htm

form in .1M PO4 works well for most TEM & SEM. A few minutes centrifugation

at low speed (200rpm) hasn't damaged my samples. It helps to cut the

proboscis off if you are not allowed to bisect the head. Standard EmBed

resin has worked well for me--just allow infiltration overnight. Proper

orientation can be the most difficult often requiring two or more

embeddings, reorienting each time. SEM prep is very similar though I have

found that critical point drying may require extended CO2 soaking times (30

to 60 minutes X 3). Some of our mutants have very little tissue in the head

and are very susceptible to drying artifacts (they collapse). Some people

leave the head attached to the body for ease of handling. I don't. Use low

accelerating voltages in the SEM (10 kV or less) to minimize charging, etc.

Good luck!

Larry D. Ackerman (415) 476-8751

Howard Hughes Medical Institute FAX (415) 476-5774

UCSF, Box 0724, Rm U426

533 Parnassus Ave. mishot@itsa.ucsf.edu

San Francisco, CA 94143

First, for the TEM part: if you can get newly emerged adults, the cuticle

will be soft and reasonably easy to section. By "newly emerged" I mean

within a few hours. After the heads have assumed their normal shape (they

inflate to rupture the pupal exoskeleton), but before they have tanned

significantly. This assumes that the client isn't investigating something

in the eye that changes subtly with tanning, or age after emergence.

The most difficult part of the eye for TEM are the crystalline cones. If

the client isn't interested in them for TEM, dissect away the exoskeleton

(cuticular area over the eye) and the underlaying c. cones. If possible,

before fixation or embedding. (Waiting for the laughter to die here.) The

easiest way to do this is chopping away with a glass knife after the blocks

are ready.

You can handle the retinal tissues pretty much like any neural tissue. The

client should already have the relevant fixation references, but routine

fixation should be pretty normal. I used pH 7.2, 0.1-0.15 M buffers for

freshwater crustaceans, standard Karnovsky's.

If s/he does want the cuticular and crystalline lenses ... wait for the

references, or a response from a _Drosophila_ / small insect specialist--I

worked on crustaceans (there are differences in the cuticle).

Use a *hard* resin with low viscosity. Other than that, I've seen no

advantage for one type over another; except what works for you.

The heads should sink, if they don't, a *little* spinning won't hurt. By

hand, I wouldn't use a centrifuge for this, even on the lowest speed. Mild

vacuum would be better.

You mention "heads", so: is that what the client is bringing you, or are

you planning on decapitating the critters? This would be good, if you can

find a guillotine small enough. Best would be to then bisect the heads

midsaggitally, if you can handle (orient, etc.) the resulting hemiheads.

They may be pretty difficult to see to orient after OsO4, although the

cuticle won't take up much osmium.

For SEM: treat like for TEM, except leaving the heads intact. CPD works

well, and drying from HMDS can also (HMDS was originally used for

Malphigian tubules in insects). Crystalline cones can be exposed nicely for

SEM by dry-fracturing the heads after drying and mounting on the stubs.

Read: hitting the eye with a razor blade, then gently blowing away the

debris with a duster. (Also, look in through the back of an intact head.)

Mount on double-stickey *carbon conductive* tape, then run a thin line of

silver paint to the head. Touch the wet paint to a sacrificial area of the

head (one you don't care about), and draw a ring of Ag paint around the

head, as close as you can get without touching it.

All of this last is to insure maximum conductivity of the specimen. The

setae will charge like buggers otherwise, and you'll have bright little

hairs, and lose structural details of both the setae and surrounding

cuticle. Make certain that the heads have both excellent mechanical and

electrical contact to the stub.

oshel@staff.uiuc.edu