Louise,

You might find that your control blocks, like most lab's control tissue, are
from old tissues that may have been stored in formalin for several weeks
prior to being processed as control blocks. Your test cases are usually
processed after the minimum of fixation (possible 6hrs to 1-2 days).
Studies show that many antigens are masked by formalin and this
masking increases with fixation time. Antigen retrieval on blocks fixed for
long periods often needs to be longer than those fixed for shorter, more
routine times.

Eg I recently prepared blocks for a CMV control from liver that had been
collected 2 years ago. IPX staining was positive but weaker than other
test cases and weaker than the original control slides that had been
used previously. This may be a good thing. If you can demonstrate CMV
in the control, than you are giving the test case every chance to stain if
CMV is there. ie your control is your lower limit of detection.

This may not work for your thyroid control. You may need to obtain
thyroid blocks from recently collected tissue and process them. The
fixation time should be similar to that of your test cases.

Hope this helps,

Tony
Tony Henwood JP, BappSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager
The Children's Hospital Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: (02) 9845 3306
Fax: (02) 9845 3318

>>> Louise Taylor <louiset@mail.saimr.wits.ac.za> 14/February/2001
08:03pm >>>
Hi all

This is a desperate plea to anyone out there that has woked with the
antibody CATHEPSIN B
The specification sheet recomends using thyroid as a control
I find that I cannot get the control block and the test cases to work at the
same dilutions
The controls are positive at 1:100
The test cases are positive at 1:400 and 1:800
when doing the test cases at 1:100 there is too much background
staining
The protocol followed is HIER WITH CITRATE BUFFER ATpH 6
Overnight incubation with primary ab
Followed with biotinylated anti-sheep at 1:200
Then Vector ABC

Research Laboratory
Department of Anatomical Pathology
South African Institute for Medical Research
Johannesburg
South Africa