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From:bbracing <> (by way of histonet)
To:histonet <>
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ER/PR proteins are somewhat soluble in alcohol based fixatives, so you will
either lose them or they will leach out into the cytoplasm.  The only way
to avoid this is to use formalin as the primary fixative.  We routinely
process fine needle aspirates and occasionally cytology specimens with
excellent results.  For our fine needle aspirates, these are always air
dried after the slides are made, then fixed for 20/30 min in 10% formalin,
then post fixed in cold (+5 to -5 C ) methanol for 6 min, then transfered
to cold acetone for 3 min., then placed in an equal mixture of saline and
glycerin, then stored in a -70C freezer untill required for
staining.Cytology specimens should be collected without fixative, the
smears made, air dried, and the same procedure undertaken. It is also
possible with cytology specimens to collect them in 10% formalin and fix
them over night, then make slides, and or cell blocks, which can be
processed in the usuall maner and stained in the usuall maner.  Once the
specimens have been adequately fixed in formalin the ER/PR roteins are no
longer affected by alcohol. This is why routine surgical specimens can be
processed without loss of ER/PR reactivity.  With the air dryed method, no
heat induced antigen retrieval is needed, with the latter it is necessry.
We use DAKO antibodies, with a one hour incubation in the primary antibody,
30 min in the link and lable reagents, a 10 min treatment with DAB, a 1
min. heamatoxylin counter stain which is always differentiated in 0.5% HCl
in 70% alcohol to clear the nuclie for better sensitivity, blued in lithium
carbonate, dehydrated and cleared and mounted in the usuall manner.   I
hope that this is of some help
                                           Kerry Beebe  (ART)     e-mail
                                           Kelowna Gen Hosp
                                           Kelowna B.C.  Canada

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