sticky slides /overfixation
|From:||RUSS ALLISON <Allison@Cardiff.ac.uk>|
Here comes a blast from the past!
I forever remember the words of Edward Brain ( Royal College of
Surgeons of England), That a common cause of decalcified
sections floating off slides during staining was incomplete
dehydration during processing. I believe he was right.
I think that may also apply to immuno staining; i.e. incomplete
processing dehydration = loss of sections during staining.
The concept of "overfixation" is in some ways related.
What happens during fixation depends upon the fixing agent
employed. Alcohol, for example can - and does - fix completely in
that it is a precipitant (alters the water balance and brings things -
essentially proteins - out of solution).
If we are talking about "formalin", then the term "overfixation" is
much harder to comprehend for we know that the "cross-linking"
which formaldehyde facilitates can continue for days, nay, weeks,
nay, months, nay years! Truly. The concept of overfixation can
therefore be seen to be a rather arbitrary concept.
It is likely, very likely in my opinion, that at some point the degree
of cross linking becomes suffieicient to hinder access of larger
molecules, of which antibodies are an extremely good example.
Therefore, fixation for immunocytochemistry needs to be optimised,
but that is easier said than done, for few tissues are homogenous.
Fortunately, there is a degree of tolerance and that is what we
seek to establish.
"Antigen retreival" seeks to reverse the effects of cross-linking,
again with a reasonable degree of tolerance, although we try very
hard to standardise that!
Remember, however, the aims of fixation are to inhibit - prevent -
autolysis and putrefaction. In a nut shell, that means killing bugs
and inactivating enzymes. Easy peasy!
Rip van Winkle is now going back to sleep
Good night all!
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