Here I am, behind this bush over here...

I've posted Annie (in Africa)'s message below, as published in Microscopy
Today, May '98.

Thanks for the article Annie! You are getting MT are you not?

Phil

Hints for fixing vertebrate eyes:
        In my experience (up to 6 eyes per week for about 10 years), the
globes MUST be properly fixed. I learned this the hard way, when I was a
Medical Technologist, in charge of the Neuropathology Laboratory at the
University of Cape Town Medical School, Anatomical Pathology Department.
        We would 'lop' off the 'top' and the 'bottom' - like a breakfast
soft boiled egg - after 24 hours in 10% buffered formalin. The cuts are
made in line with the cornea after orienting the specimen properly. The
inferior and superior pieces were processed routinely overnight if properly
fixed - with excellent results.
        The roughly 8mm thick mid-portion should be handled according to
the contents of the globe - sometimes another 24 to 48 hours fixation is
necessary for tumor or blood filled globes - particularly if the tumor is
necrotic.
        Thereafter a long dehydration program is required:
        3 changes of absolute alcohol, total time about 8 or 10 hours
        follow with a manual inspection to check for proper dehydration
        6 to 8 hours total in 3 changes of 100 % xylene (or other clearing
agent)
        ca . 12 hours total in 3 wax changes
        embed routinely in paraffin wax.
        When sectioning use lots  of ice and section at 4 to 6 micrometers.
I used to float out on 10% alcohol (or weaker) before the warm water bath.
My results were excellent, with very few eyes giving me problems. I
have used the double embedding method as well (to teach students) and have
found it to be a poor substitute for the careful, slow processing of eyes
using the classic pathway.
        I have to admit that many of my colleges did not enjoy handling the
eyes, claiming that they could not get the results which I got. I had many
years of practice and an excellent teacher. In addition, my Pathologist had
"an understanding" of the lengthy schedule and he in turn educated the
ophthalmic surgeons and the oncologists to be patient if they wanted good
results.

Anne S. van Binsbergen, South African Institute for Medical Research,
Chris Hani Baragwanath Hospital in Soweto, South Africa

>Hello Histonetters,
>Philip Oshel - somewhere out there - has a protocol for whole eye
>processing which I e-mailed him about a year or so ago, for ? pulication in
>Microscopy today.
>Philip, my system 'crashed' and I was out of action for ages - lost all my
>e-mail info (sadly) - please would you re-post that protocol - if you have
>it still!
>
>I found that pig eyes were a fairly good substitute for human eyes.
>
>The secret to proper processing of multi-layered tissues is 'slowly and
>gently'!
>
>Regards,
>
>Annie (in Africa)
>
>Anne S. van Binsbergen
>T.I.C. Anatomical Pathology
>S.A.I.M.R.
>Chris Hani Baragwanath Hospital
>PO Box 1038
>Johannesburg 2000
>South Africa
>
>tel: 011 4898711
>cell: 083 4403343

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Philip Oshel
Technical Editor, Microscopy Today
PO Box 620068
Middleton, WI  53562
Voice: (608) 833-2885
Fax: (608) 836-1969 (please make sure my name is on any fax)
oshel@terracom.net