[Histonet] IHC Slides, Hotplate vs Oven

[Tony Henwood]

Eric,

Some of my thoughts on your discussion questions:

1. How do you monitor the shortened baking time, especially when
multiple racks of slides are cut throughout the day?
We have timers set for when each rack goes into the oven. This might be
an issue for labs with larger batches (we are a Children's hospital)

 2. Assuming charged slides and cell conditioning/antigen retrieval are
employed, are you seeing major problems with section adhesion?
No, not usually. Problem sections tend to be those that are inadequately
fixed (& therefore inadequately processed), brain and bone sections.
They may tend to lift.

 3. What if slides are received from another institution for staining,
or slides get baked longer than 25 mins? 
Again, it is difficult to know the conditions that the tissues have
undergone. In-built controls, if present are invaluable.
We use a BondMax immunostainer, so the antigen retrieval temperature and
time are better controlled than is possible with the manual procedure
(or when I do it!).

I believe that incomplete fixation causes more problems for morphology
and immunohistochemistry than is commonly appreciated. 

"Fix the fixing and most problems are solved" - Hows that for a sweeping
statement!

Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead 
Cnr Hawkesbury Road and Hainsworth Street, Westmead 
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 




-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Gagnon,
Eric
Sent: Thursday, 22 April 2010 5:31 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] IHC Slides, Hotplate vs Oven


Tony's answer (62 degrees C for 25 mins) is the minimum to ensure
section adheres to slide.  We all have to stain slides that have been
baked longer than 30 mins.  However, especially for Her2, longer baking
times can result in false negatives or at least diminished staining.
The justification for baking longer is an attempt to prevent the section
washing off or lifting.  We are seeing evidence that section adhesion is
not a good tradeoff for diminished staining.  Ideally both can be
achieved with no tradeoff.
 
Tony or others who may wish to answer:
 
1. How do you monitor the shortened baking time, especially when
multiple racks of slides are cut throughout the day? 2. Assuming charged
slides and cell conditioning/antigen retrieval are employed, are you
seeing major problems with section adhesion? 3. What if slides are
received from another institution for staining, or slides get baked
longer than 25 mins?
 
Thanks in advance,
 
Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada
 
 


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