[Histonet] Flippin on the Bond

[Tony Henwood (SCHN) via Histonet]

Hi all,

I was reading an article entitled “Evaluation of Tissue Adhesion and Staining 
Performance of BOND Adhesive Microscope Slides: A Comparative Study on the 
BOND-III and BOND-MAX Platforms” 
(https://pdfs.semanticscholar.org/f5f1/ad0741bc5445d0e1079a0d07a32c8a3a26c1.pdf)
 and they make mention of the “Flippin Protocol”, an amended protocol widely 
used by BOND customers.
It is used for those antibodies that are adversely affected by the endogenous 
peroxidase block that is used in the Bond kits (and possibly with other 
non-Bond kits as well).
In this protocol, the peroxidase block is applied after the localisation 
antibody is applied. Examples given by Leica are PAX5 and AMACR.

The paper refers to several other antibodies as well (CD3, CD34, SMA and TTF1) 
that uses the “Flippin protocol”. Now I have not had issues with the first 
three antibodies (they work quite well with the standard protocol, ie 
peroxidase block before the localisation antibody) and have no experience with 
TTF1 (we are a pediatric laboratory).

Has anyone experienced this issue with antibodies and use the Flippin protocol?

What antibodies are an issue?

Thanks for your input

Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) | Principal 
Scientist; Adjunct Fellow, School of Medicine, University of Western Sydney; 
Visiting Lecturer, School of Life Sciences, Faculty of Science, University of 
Technology Sydney | Histopathology
t: (02) 9845 3306 | e: 
tony.henw...@health.nsw.gov.au | w: 
www.schn.health.nsw.gov.au
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Re: [Histonet] Frozen section slides fading

[Tony Henwood (SCHN) via Histonet]

Hi Dane,

Fading and especially leaching of the eosin, will occur if slides are 
incompletely dehydrated. Flip out the subsidiary condenser lens (or drop the 
condenser) and look for refractile water droplets.
Certain mountants can also cause fading of H
I would also be concerned about the xylene substitute.

Alternatively, after staining and ethanol dehydration, air dry the slides 
(making sure they are completely dry) and directly coverslip with your mountant 
(without the substitute added). This will reduce the risk of xylene exposure.

Hopefully this is useful


Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-Original Message-
From: Hill, Dane via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, 17 March 2020 1:01 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Frozen section slides fading


Hello all,


Thank you, in advance, for your help and for the information you've already 
posted online. It is helpful. I am hoping you can help me troubleshoot an issue 
we are having. I am the Mohs fellow at Texas Tech dermatology. For the last 9 
months or so we have been noticing that the longer our frozen section slides 
are away from the day of surgery, the more they develop these pink, amorphous 
spots in certain areas of the tissue. The further away from the surgery, the 
more faded they get. The actual tissue remains intact on the slide, with 
complete epidermis evaluable, but it's almost as if all the hematoxylin fades 
or something because it just turns into that bright pink color, but does not 
hold any of the purple. We have tried changing our xylene substitutes and even 
changed the hematoxylin and eosin stains. The thinner (5 micron cuts) are 
affected long before the 20 micron cuts. Photos were too big to be attached, 
but I can PM someone with them, if needed. They are from slides prepared just a 
few weeks ago and are just starting to fade. In about 6 months they will turn 
all pink.

Interestingly, this is not happening on any of our permanent sections. They 
remain perfectly stained for years. And it will happen, even if we don't "bake" 
any of the slides or heat them in storage. The only thing the techs say they do 
differently for the frozen sections is use xylene substitute in place of 
xylene. They also mix a little xylene substitute in the glue to cover slip the 
slides. I wasn't sure if that could be contributing. If there are any testing 
protocols of specific reagents we use or adjustments in staining that have 
previously remedied the issue, I'd be very grateful as we have been 
unsuccessful at pinning it down. Thank you, again!

Dane Hill


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[Histonet] clearing agents

[Lowen, Nancy via Histonet]

Good Morning,
Was wondering what preferences are as to CitriSolv versus Clearite 3 or Propar 
3?
Process lots of bone tissue and wondering which one everyone thinks works best.
Thanks
Nancy
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[Histonet] Tissue Blocks

[Ken M via Histonet]

Hello All:

I have thousands of tissue blocks from a large US certified reference lab. Most 
of them were used for IHC diagnosis. I am looking to trade what I have for more 
common Histology blocks for control slides such as Mast Cell, Pneumocystis, 
Copper, Gram, Aspergillus fungus, H-Pylori, Spirochete, Acid Fast Bacteria and 
other common Histology tissue for special stain control slides. I also have 
lots of normal tissue blocks. Please let me know if you are interested.

Ken Marzinsky
4122 Bennett Memorial Road
Durham, NC 27712
713-822-0256
kdea...@hotmail.com
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[Histonet] Frozen section slides fading

[Hill, Dane via Histonet]

Hello all,


Thank you, in advance, for your help and for the information you've already 
posted online. It is helpful. I am hoping you can help me troubleshoot an issue 
we are having. I am the Mohs fellow at Texas Tech dermatology. For the last 9 
months or so we have been noticing that the longer our frozen section slides 
are away from the day of surgery, the more they develop these pink, amorphous 
spots in certain areas of the tissue. The further away from the surgery, the 
more faded they get. The actual tissue remains intact on the slide, with 
complete epidermis evaluable, but it's almost as if all the hematoxylin fades 
or something because it just turns into that bright pink color, but does not 
hold any of the purple. We have tried changing our xylene substitutes and even 
changed the hematoxylin and eosin stains. The thinner (5 micron cuts) are 
affected long before the 20 micron cuts. Photos were too big to be attached, 
but I can PM someone with them, if needed. They are from slides prepared just a 
few weeks ago and are just starting to fade. In about 6 months they will turn 
all pink.

Interestingly, this is not happening on any of our permanent sections. They 
remain perfectly stained for years. And it will happen, even if we don't "bake" 
any of the slides or heat them in storage. The only thing the techs say they do 
differently for the frozen sections is use xylene substitute in place of 
xylene. They also mix a little xylene substitute in the glue to cover slip the 
slides. I wasn't sure if that could be contributing. If there are any testing 
protocols of specific reagents we use or adjustments in staining that have 
previously remedied the issue, I'd be very grateful as we have been 
unsuccessful at pinning it down. Thank you, again!

Dane Hill


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