whole eye processing protocol
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
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
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
+2711 4898717 (fax)
+2783 2526252 (cell)
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