Re: [Histonet] Atherosclerosis and freshly cut tissue

From:"Anila Syed"

Hi Ross,
Thank you very much for your reply.
The trouble is that I need to collect a sufficient quantity to give to my
pathology department to put into their machine. How do people usually get
around this problem of tiem in formalin? The machine is on a three day cycle
and they wont run through a small number of tissue samples for me, they will
only do it if there are enough to fill one bucket.

Would you say  manual processing is a viable option?

----- Original Message -----
From: "Stapf, Ross" 
To: "Anila Syed" ;

Sent: Wednesday, August 25, 2004 9:05 PM
Subject: RE: [Histonet] Atherosclerosis and freshly cut tissue

1. More than 48 to 72 hours in formalin can cause big problems for
immunos in my experience.

2. My former lab had major problems with certain antibodies when slides
were cut in advance and we cut those antibodies fresh, or had to use
certain others within 2 weeks.  Slides to be stained with other
antibodies were good for months.  It was a case of experience.  When we
had a problem we found that the same tissue block stained fresh was much
better than when it had been cut a month or sometimes a month before.
For other antibodies there was no difference.

Here we are able to cut everything ahead, but we rarely cut more than 10
slides per antibody ahead.  Some of those are refrigerated to slow down
the loss of antigenicity.

My advice is that if someone told you to cut them fresh and if they have
worked with the antibody you are dealing with or at least do immuno's
reguarly in your institution, then trust their word.  The need to cut
fresh seems to vary some by climate and fixation/processing protocal as
well as antibody.  Refrigeration or freezing of the slides may help you
to store them longer.

I would be much more worried about your fixation times.  2 months is a
long time in formalin.

Ross M Stapf
Histopathology Manager
Baylor University Medical Center
3500 Gaston Ave.
Dallas, TX 75246
214-820-4110 fax

-----Original Message-----
[] On Behalf Of Anila
Sent: Wednesday, August 25, 2004 2:38 PM
Subject: [Histonet] Atherosclerosis and freshly cut tissue

Dear All,

We are currently collecting carotid endarterectomies in order to stain
them for the presence of inflammatory markers and various endothelial
receptors. We collect the tissue into 10% formalin and it has been left
like that for about two months while we gather sufficient numbers.

My question is, we have been told that once they are wax embedded, they
will need to be freshly cut each time in order for the IHC to work. Is
this true?

Could I cut a batch of, say 50 sections and keep them until they need to
be stained? Or will I need to cut fresh sections each time. I want to
stain for MMP's, TIMP1 etc.

Also, in general, is it true that receptor proteins start to become
oxidised in wax-embedded tissue and therefore staining starts to

I hope someone has some experience of this.

Thanks in advance for your help.

Best wishes

Anila Syed

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