[Histonet] RE: immunohistochemistry help please

[Tony Henwood (SCHN)]

Rebecca,

Try making sure the sectioned slides have been adequately dried (up to 65oC for 
at least 30 minutes) before de-waxing.
I believe that much of the de-waxing of sections occurs during the slide drying 
phase and this is not given the importance it deserves.
You could also try heated detergent dewaxing:
Henwood A (2012) "The application of heated detergent dewaxing and rehydration 
to immunohistochemistry" Biotechnic & Histochemistry 87(1):46-50.
Henwood, A. F., Prasad, L., & Bourke, V. M. (2013). "The application of heated 
detergent dewaxing and rehydration to techniques for the demonstration of 
fungi: a comparison to routine xylene-alcohol dewaxing" J Histotechnol 
36(2):45-50.


Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
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-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Tarantelli, 
Rebecca Anne
Sent: Saturday, 3 August 2013 12:30 AM
To: Sebree Linda A; histonet@lists.utsouthwestern.edu
Subject: [Histonet] RE: immunohistochemistry help please

Thanks Linda, 

This is going to be a very dumb question (may I remind you this is my very 
first IHC experience) how do I know the melting point of my paraffin? It was 
not a new bottle of xylene, and the tissues are all from the same block cut 
into sequential slides 


Rebecca 


-Original Message-
From: Sebree Linda A [mailto:lseb...@uwhealth.org] 
Sent: Thursday, August 01, 2013 3:50 PM
To: Tarantelli, Rebecca Anne; histonet@lists.utsouthwestern.edu
Subject: RE: immunohistochemistry help please

OK, so I would be very suspect of your xylene.  Has anything changed, i.e. 
vendor?  It sounds like it's not dissolving your paraffin.  Are you using the 
same paraffin you were when you were successful?  Do you dry/melt your slides 
in an oven prior to depar?  If not, you could try that at a  temperature just 
above the melting point of your paraffin for 10 - 30 minutes.

Linda A. Sebree
University of Wisconsin Hospital & Clinics IHC/ISH Laboratory
600 Highland Ave. 
Madison, WI 53792
(608)265-6596
FAX: (608)262-7174 


-Original Message-
From: Tarantelli, Rebecca Anne [mailto:ra...@pitt.edu]
Sent: Thursday, August 01, 2013 2:40 PM
To: Sebree Linda A; histonet@lists.utsouthwestern.edu
Subject: RE: immunohistochemistry help please

Hi Linda, 

I'm sorry - I should have been a little more clear. Once I realized it isn't 
working, I did stop using the antibody. I have been trying just 
deparaffinization, and then counter staining with hematoxylin and skipping 
everything in between. The tissue isn't taking up the hematoxylin. With your 
experience, would you have any suggestions as to what part of the 
deparaffinization stage would cause the hematoxylin not to stain? I use: Xylene 
3 minutes x 3, 100% ethanol 3 minutes x 2, 90 % ethanol 3 minutes x 1, 75% 
ethanol 3 minutes x1, distilled water 2 minutes x 1, then Dako wash buffer 3 
minutes x 1. Next, I have been just going right to 3 minutes in my hematoxylin. 

I'm sorry if these are beginner questions, this is my first attempt at IHC, and 
it worked so well for a week and now nothing. 

Thank you!

Rebecca Tarantelli


-Original Message-
From: Sebree Linda A [mailto:lseb...@uwhealth.org]
Sent: Thursday, August 01, 2013 3:36 PM
To: Tarantelli, Rebecca Anne; histonet@lists.utsouthwestern.edu
Subject: RE: immunohistochemistry help please

To test your deparaffinization Rebecca, I'd try staining a slide with something 
cheaper and quicker, like just your counterstain to see if that's working.  
Then go from there.

Linda A. Sebree
University of Wisconsin Hospital & Clinics IHC/ISH Laboratory
600 Highland Ave. 
Madison, WI 53792
(608)265-6596
FAX: (608)262-7174 

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Tarantelli, 
Rebecca Anne
Sent: Thursday, August 01, 2013 2:20 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] immunohistochemistry help please

Hi all,

I am new to this forum, but a friend recommended I send my issue out to you for 
help. Four weeks ago I completed a new protocol for Immunohistochemistry (IHC) 
using protinase K epitope retrieval, and monoclonal antibody 3F6. It worked 
perfectly. Three days later, after 13 beautiful stains, it just stopped 
working. The slides will not even take up the counter stain - Hematoxylin. I 
have exchanged my xylene, and tried again, and it still is not working. I am 
guessing since my counter stain isn't working, this is an issue in the 
deparaffinization stage, but I'm just not sure.

Please let me know if you have any suggestions!

Thank you
Rebecca

Rebecca Tarantelli

RE: [Histonet] Portable fans in the lab

[Sanders, Jeanine (CDC/OID/NCEZID)]

We put freshly cut slides on the stainer regularly and we use charged slides 
exclusively. I simply take each slide and give each a quick sharp flick to snap 
the excess water out from under the section as I place them in the staining 
rack and then load on the stainer.

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Victoria Baker
Sent: Sunday, August 04, 2013 12:32 PM
To: Rene J Buesa
Cc: histonet@lists.utsouthwestern.edu; Jeanne Clark
Subject: Re: [Histonet] Portable fans in the lab

Hi
Rene is correct about standing them up right to drain, but if you are using 
charged slides water gets trapped underneath the sections because of the + 
charge of the slide itself.  If you see a bubble on the last section or around  
sections on a ribbon you can release the water by just pin pricking them on the 
edge with a needle point or the tip of a fine tipped forceps. Tap the section 
and the water will drain away from the tissue.
Joelle is on target with use of a fan if your lab has an increased or high 
humidity level as this will also affect the water retention on a slide.






On Sun, Aug 4, 2013 at 11:50 AM, Rene J Buesa  wrote:

> In my experience the best procedure is to place the slides with the 
> sections just "fished" from the water bath in a vertical position on 
> the short edge (1 plg).
> By doing so the water that may be under the section will come out and 
> after about 5 minutes the slides can be placed in the rack and either 
> go to the oven to dry before manual staining or directly to the 
> autostainer heating station.
> If you blow air over a section that is in horizontal position any 
> water trapped under the section will remain there and can cause the 
> artifact known as "nuclear bubbling".
> René J.
>
>
> 
> From: Jeanne Clark 
> To: "histonet@lists.utsouthwestern.edu" 
>  >
> Sent: Saturday, August 3, 2013 5:34 PM
> Subject: [Histonet] Portable fans in the lab
>
>
> Anyone using portable fans in Histology to facilitae drying of parafin 
> sections on slides prior to drying in oven?
>
> Jeanne
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RE: [Histonet] Air bubbles in Coverslipper

[Sanders, Jeanine (CDC/OID/NCEZID)]

we often have to change the dispensing nozzle when this occurs.

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Anna Huntley 
Coffey
Sent: Friday, August 02, 2013 3:56 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Air bubbles in Coverslipper

Hi Histonetters!

We have a Leica CV5030 coverslipper in our lab and we regularly (~ once a week 
or so) have trouble with large air bubbles showing up in a significant number 
of our slides.  We have flushed the hose from the mounting medium through the 
needle with xylenes which sometimes works to clear the bubbles and sometimes 
does not.  Has anyone else had trouble with this and possibly found a solution? 
 I would really appreciate your advice!

Thanks,
Anna
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[Histonet] Re: Histonet Digest, Vol 117, Issue 6

[ewj]

as long as they are not Cubs fans there should be 
no real problem, imho.



>  ---Original Message---
>  From: histonet-requ...@lists.utsouthwestern.edu
>  To: histonet@lists.utsouthwestern.edu
>  Subject: Histonet Digest, Vol 117, Issue 6
>  Sent: Aug 05 '13 01:00
>  
>  Send Histonet mailing list submissions to
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>  
>  When replying, please edit your Subject line so it is more specific
>  than "Re: Contents of Histonet digest..."
>  Today's Topics:
>  
>     1. Portable fans in the lab (Jeanne Clark)
>     2. RE: Portable fans in the lab (joelle weaver)
>     3. Re: Portable fans in the lab (Rene J Buesa)
>     4. Re: Portable fans in the lab (Victoria Baker)
>     5. Portable fans in lab (Jeanne Clark)
>  From: Jeanne Clark 
>  To: "histonet@lists.utsouthwestern.edu" 
>  Date: Sat, 3 Aug 2013 14:34:28 -0700 (PDT)
>  Subject: [Histonet] Portable fans in the lab
>  
>  Anyone using portable fans in Histology to facilitae drying of parafin 
> sections on slides prior to drying in oven?
>   
>  Jeanne
>  From: joelle weaver 
>  To: Jeanne Clark ,
>  "histonet@lists.utsouthwestern.edu" 
>  Date: Sun, 4 Aug 2013 10:58:55 +
>  Subject: RE: [Histonet] Portable fans in the lab
>  
>  Yes, small fan to dry slides after cover slipping ( done by hand)
>  
>  
>  
>  
>  Joelle Weaver MAOM, HTL (ASCP) QIHC
>  
>  > Date: Sat, 3 Aug 2013 14:34:28 -0700
>  > From: paintedsplas...@yahoo.com
>  > To: histonet@lists.utsouthwestern.edu
>  > Subject: [Histonet] Portable fans in the lab
>  >
>  > Anyone using portable fans in Histology to facilitae drying of parafin 
> sections on slides prior to drying in oven?
>  >  
>  > Jeanne
>  > ___
>  > Histonet mailing list
>  > Histonet@lists.utsouthwestern.edu
>  > http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>         
>  From: Rene J Buesa 
>  To: Jeanne Clark ,
>  "histonet@lists.utsouthwestern.edu" 
>  Date: Sun, 4 Aug 2013 08:50:42 -0700 (PDT)
>  Subject: Re: [Histonet] Portable fans in the lab
>  
>  In my experience the best procedure is to place the slides with the sections 
> just "fished" from the water bath in a vertical position on the short edge (1 
> plg).
>  By doing so the water that may be under the section will come out and after 
> about 5 minutes the slides can be placed in the rack and either go to the 
> oven to dry before manual staining or directly to the autostainer heating 
> station.
>  If you blow air over a section that is in horizontal position any water 
> trapped under the section will remain there and can cause the artifact known 
> as "nuclear bubbling".
>  René J.
>  
>  
>  
>  From: Jeanne Clark 
>  To: "histonet@lists.utsouthwestern.edu" 
>  Sent: Saturday, August 3, 2013 5:34 PM
>  Subject: [Histonet] Portable fans in the lab
>  
>  
>  Anyone using portable fans in Histology to facilitae drying of parafin 
> sections on slides prior to drying in oven?
>   
>  Jeanne
>  ___
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>  Histonet@lists.utsouthwestern.edu
>  http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>  From: Victoria Baker 
>  To: Rene J Buesa 
>  Date: Sun, 4 Aug 2013 12:31:40 -0400
>  Subject: Re: [Histonet] Portable fans in the lab
>  
>  Hi
>  Rene is correct about standing them up right to drain, but if you are using
>  charged slides water gets trapped underneath the sections because of the +
>  charge of the slide itself.  If you see a bubble on the last section or
>  around  sections on a ribbon you can release the water by just pin pricking
>  them on the edge with a needle point or the tip of a fine
>  tipped forceps. Tap the section and the water will drain away from the
>  tissue.
>  Joelle is on target with use of a fan if your lab has an increased or high
>  humidity level as this will also affect the water retention on a slide.
>  
>  
>  
>  
>  
>  
>  On Sun, Aug 4, 2013 at 11:50 AM, Rene J Buesa  wrote:
>  
>  > In my experience the best procedure is to place the slides with the
>  > sections just "fished" from the water bath in a vertical position on the
>  > short edge (1 plg).
>  > By doing so the water that may be under the section will come out and
>  > after about 5 minutes the slides can be placed in the rack and either go to
>  > the oven to dry before manual staining or directly to the autostainer
>  > heating station.
>  > If you blow air over a section that is in horizontal position any water
>  > trapped under the section will remain there and can cause the artifact
>  > known as "nuclear bubblin

[Histonet] Re: Portable fans in the lab

[Bob Richmond]

I've attempted to use a portable fan to dispel formaldehyde fumes in a lab
with no effective ventilation (not required in Tennessee) and was told by
management they were prohibited. OSHA seems not to understand that
pathologists and histologists are people too.

Bob Richmond
Samurai Pathologist
Maryville TN
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[Histonet] Parts for VIP 2000 processor

[MaryK Mendell]

Does anyone know where I can get parts for  a VIP2000, need a gasket retort 
seal and gasket solution reservoir seals.  Thank-you in advance



Kate




Kate Mendell
Histopathology/Lab Manager

Anyone who stops learning is old, whether at twenty or eighty.  ~Henry Ford

HOWARD S. GOLDBERG, M.D., INC
990 Paradise Road
Swampscott, MA  01907
TEL:  781.595.0151
FAX:  781.592.6780
kmend...@goldbergmd.net
www.cosmesticdermcenter.com
PRIVACY NOTICE: This e-mail message may contain confidential patient or other 
information belonging to the sender that is legally privileged.  This 
information is intended only for the use of the individual or authorized entity 
named above. The authorized recipient of this patient or other confidential 
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If you have received this message in error, please notify the sender 
immediately and delete.  Please keep any information you may have viewed 
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[Histonet] Portable fans in lab

[Jeanne Clark]

One more question...any problems with compliance from Joint Commission or CMS 
on having portable fans in the lab?  As in possible health hazard?
 
Jeanne
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Re: [Histonet] Portable fans in the lab

[Victoria Baker]

Hi
Rene is correct about standing them up right to drain, but if you are using
charged slides water gets trapped underneath the sections because of the +
charge of the slide itself.  If you see a bubble on the last section or
around  sections on a ribbon you can release the water by just pin pricking
them on the edge with a needle point or the tip of a fine
tipped forceps. Tap the section and the water will drain away from the
tissue.
Joelle is on target with use of a fan if your lab has an increased or high
humidity level as this will also affect the water retention on a slide.






On Sun, Aug 4, 2013 at 11:50 AM, Rene J Buesa  wrote:

> In my experience the best procedure is to place the slides with the
> sections just "fished" from the water bath in a vertical position on the
> short edge (1 plg).
> By doing so the water that may be under the section will come out and
> after about 5 minutes the slides can be placed in the rack and either go to
> the oven to dry before manual staining or directly to the autostainer
> heating station.
> If you blow air over a section that is in horizontal position any water
> trapped under the section will remain there and can cause the artifact
> known as "nuclear bubbling".
> René J.
>
>
> 
> From: Jeanne Clark 
> To: "histonet@lists.utsouthwestern.edu"  >
> Sent: Saturday, August 3, 2013 5:34 PM
> Subject: [Histonet] Portable fans in the lab
>
>
> Anyone using portable fans in Histology to facilitae drying of parafin
> sections on slides prior to drying in oven?
>
> Jeanne
> ___
> Histonet mailing list
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> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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>
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Re: [Histonet] Portable fans in the lab

[Rene J Buesa]

In my experience the best procedure is to place the slides with the sections 
just "fished" from the water bath in a vertical position on the short edge (1 
plg). 
By doing so the water that may be under the section will come out and after 
about 5 minutes the slides can be placed in the rack and either go to the oven 
to dry before manual staining or directly to the autostainer heating station.
If you blow air over a section that is in horizontal position any water trapped 
under the section will remain there and can cause the artifact known as 
"nuclear bubbling".
René J.



From: Jeanne Clark 
To: "histonet@lists.utsouthwestern.edu"  
Sent: Saturday, August 3, 2013 5:34 PM
Subject: [Histonet] Portable fans in the lab


Anyone using portable fans in Histology to facilitae drying of parafin sections 
on slides prior to drying in oven?
 
Jeanne
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RE: [Histonet] Portable fans in the lab

[joelle weaver]

Yes, small fan to dry slides after cover slipping ( done by hand) 




Joelle Weaver MAOM, HTL (ASCP) QIHC
 
> Date: Sat, 3 Aug 2013 14:34:28 -0700
> From: paintedsplas...@yahoo.com
> To: histonet@lists.utsouthwestern.edu
> Subject: [Histonet] Portable fans in the lab
> 
> Anyone using portable fans in Histology to facilitae drying of parafin 
> sections on slides prior to drying in oven?
>  
> Jeanne
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