whole eye processing protocol

From:A van Binsbergen

Whole eye processing - Annie's Method

Fix in 10%buffered formalin overnight
Orientate correctly and very carefully horizontally cut the top (superior)
and bottom (inferior) portions off - almost like carefully slicing off the
top of a hard boiled breakfast egg!
The thicker section in the middle should be roughly 15mm thick (depends on
source of eye - human,pig, rabbit) and contain the optic nerve stump.
I would process the inf & sup pieces according to the 'contents' of the
eye - tumour (retiniblastoma) or blood filled pieces would be processed with
the mid portion.
I would process the mid portion routinely (overnight) if there were no
tumour or blood

Long Processing as follows:
Fix whole eye - 24hrs - 10% NBF  negotiate with surgeon to receive whole
eyes on a Thursday and fix overnight
Dissect on a Friday

Dissected portions:
10% NBF   24hrs  over a weekend
decalcify at this stage if required (kristensens worked for me)
50% Alcohol  2hrs  monday
70% Alcohol  6hrs  monday
96% Alcohol  overnight monday
Absolute alcohol 1st 2hrs  tues
Absolute alcohol 2nd 6hrs  tues
Absolute alcohol 3rd overnight tues
Xylene 1st   2hrs  wed
Xylene 2nd   6hrs  wed
Xylene 3rd   overnight wed
Wax 1st   2hrs  thurs
Wax 2nd   6hrs  thurs
Wax 3rd   overnight thurs
(days of week are suggestions to fit in to a non 24hrs lab routine)

Embed and cut on a Friday - section thickness 5u
Use LOTS of ice, turn the waterbath down (layers separate on hot water),
pick up on adhesive slides, leave at 37C overnight to prevent washoffs
GO SLOWLY and do a few dummy runs first to get the technique right

Educate your surgeons to be patient and 'show off' your finished product -
how often do they get to see near-perfect cross-sections of whole eyes!
I know that this method may seem to be time consumig/tedious but in my
humble opinion it is far better to go slowly and carefully and produce
something worthwhile.
I did not have much success with reversing the process and re-processing the
'hurried'eyes which turned out to be all 'mushy' as I cut them deeper.

'Annie in Africa'

Anne S. van Binsbergen
S.A.I.M.R./Chris Hani Baragwanath Hospital
South Africa

+2711 4898717 (fax)
+2711 4898711
+2783 2526252 (cell)

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