[Histonet] More on H202 issues

[Lewis, Patrick via Histonet]

Hi Everyone

Thanks for your responses.

I am looking at cell surface markers,
Sorry I should have said.

Based on what I found out below:

Methanol is out, even though I agree that Methanol does enhance the effect of 
H202 blocking.
(I suppose I could try it to see how much/if any epitope loss there is in 
relation to H202 quenching,  At least It would help identifying false positives 
that are actually H202 artifacts.)

Also it looks like increasing the concentration of H202 is out.

As to when though,

It looks like with cell surface markers I should block after the primary, or 
even after the 2ndary?
Can I do that successfully with a HRP labeled 2ndary?

thoughts?

Patrick.


What solutions or reagents should I use to dilute hydrogen peroxide
Methanol, PBS, distilled water or saline can be used to dilute hydrogen 
peroxide. Morphology of blood smears and peroxidase-rich tissues could be 
damaged by the aqueous hydrogen peroxide solution. Therefore, methanol is a 
better choice in this case. Some cell surface markers are very sensitive to 
methanol/hydrogen peroxide quenching, reducing the staining of antigenic site, 
particularly on frozen sections. So using hydrogen peroxide in PBS is 
recommended for cell surface or membrane markers.

What concentration of hydrogen peroxide is commonly used
3% hydrogen peroxide is commonly used to block endogenous peroxidase activity. 
However, certain tissues/cells/antigen (i.e. cell surface markers such as CD4) 
can be destroyed by high concentration of hydrogen peroxide. So a lower 
concentration (0.3%) should be used.

Where  should I do hydrogen peroxide blocking during IHC procedure
The blocking can be done (1) after rehydration to water and before antigen 
retrieval, (2) after antigen retrieval and before primary antibody incubation, 
(3) after primary antibody incubation, or (4) after biotinylated secondary 
antibody incubation. For certain antigens such as CD4 and CD8, hydrogen 
peroxide blocking has detrimental effect on the epitopes, thus reduce intensity 
of antibody staining. Therefore, blocking after primary antibody or secondary 
antibody incubation is recommended.


Patrick Lewis
Research Associate II Bench
Seattle Childrens Research Institute
206-884-1115

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[Histonet] Endogenous Peroxide staining help

[Lewis, Patrick via Histonet]

Hi everyone,

I seem to have a lot of endogenous Peroxide background staining in my FFPE IHC. 
 Human tonsil tissues, with some attached muscle.

I do a H202 block at 0.3% H202 in TBS pH 8.0 for 30 minutes.

Then I wash x3  with TBST 0.05% Tween20, pH 8.0

Then I serum block with 2% NGS in TBST for 30 minutes.

Then I wash x3 TBST

Then I add my primary antibody in 2% NGS in TBST overnight.

Then I wash x3 TBST

Then I add my HRP-labeled 2ndary antibody for 30 minutes.

Then I wash x3 TBST

Then I have add my AEC Substrate.

Should I increase the concentration of H202?
Should I increase the time in H202?

Is there a different step in the IHC protocol that is better for blocking 
endogenous H202 activity?
Is it possible to lose/reverse the blocking for some reason?

I am concerned that my HRP labeled secondary may be to blame.

Thoughts?

Patrick.

Patrick Lewis
Research Associate II Bench
Seattle Childrens Research Institute
206-884-1115

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[Histonet] Cryostat vacuum

[Morken, Timothy via Histonet]

I just bought one from IMEB :

http://www.imebinc.com/



Hello Histonet,



Does anyone know where I can find a cryostat vacuum?  I've used them in the 
past but can't seem to find a manufacturer.



Thanks!



Michael J. Dessoye, M.S. | Histology/Toxicology/RIA Supervisor | Wilkes-Barre 
General Hospital | An Affiliate of Commonwealth Health | mjdessoye at 
commonwealthhealth.net>
 | 575 N. River Street | Wilkes Barre, PA 18764 | Tel: 570-552-1432 | Fax: 
570-552-1486


Tim Morken
Pathology Site Manager, Parnassus
Supervisor, Electron Microscopy/Neuromuscular Special Studies
Department of Pathology
UC San Francisco Medical Center

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[Histonet] PAP stains done by hand

[Nancy Schmitt via Histonet]

Happy Friday-

Could you please share how you are handling the potential for cross 
contamination in non-gyn pap specimens?  Are you filtering/changing out 
solutions between each case?

I appreciate your input-

Nancy
Histology Coordinator
Dubuque, IA  52001
Check us out at www.uclaccess.com





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[Histonet] Studying for ASCP qIHC

[Coffey, Anna (NIH/NCI) [C] via Histonet]

Oops! I meant answer key instead of study guide. Sorry!



Message: 4

Date: Fri, 11 Sep 2015 16:54:24 +

From: "Coffey, Anna (NIH/NCI) [C]" 
mailto:anna.cof...@nih.gov>>

To: 
"histonet@lists.utsouthwestern.edu"


mailto:histonet@lists.utsouthwestern.edu>>

Subject: [Histonet] Studying for ASCP qIHC

Message-ID: 
<5c3e10119a1b824fbe92b08279f74a91025b2...@msgb10.nih.gov>

Content-Type: text/plain; charset="us-ascii"



Hi everyone,



I've been studying for my qIHC exam at the end of this month and I bought the 
Michigan Society's study workbook which has been a very helpful study guide. 
It's my understanding that there is no accompanying study guide for this...is 
that right? If that's not true, I'd love to know where I can pick one up! If it 
is true, is there anyone (or a group) out there with an interest in developing 
one? I'd love to work on this with some folks if anyone is interested!



Best,

Anna

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[Histonet] Studying for ASCP qIHC

[Coffey, Anna (NIH/NCI) [C] via Histonet]

Hi everyone,

I've been studying for my qIHC exam at the end of this month and I bought the 
Michigan Society's study workbook which has been a very helpful study guide. 
It's my understanding that there is no accompanying study guide for this...is 
that right? If that's not true, I'd love to know where I can pick one up! If it 
is true, is there anyone (or a group) out there with an interest in developing 
one? I'd love to work on this with some folks if anyone is interested!

Best,
Anna
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[Histonet] DISPOSABLE STERILE FORCEPS

[Nirmala Srishan via Histonet]

Hi,

Does anyone know of a vendor who supplies sterile, individually wrapped , 
plastic forceps.

Thanks for the input.


Nirmala Srishan
Histology Supervisor
Holy Name Medical Center
718 Teaneck Road
Teaneck NJ 07666
Lab: 201 833 3023
Office: 201 541 6328







Holy Name Medical Center is ranked among the top hospitals in the nation 
for patient care, clinical performance and workplace excellence.
Click here to learn more.

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[Histonet] Dallas HT Position in IHC Lab

[Pat Patterson via Histonet]

HISTOTECHNICIAN (IMMUNOHISTOCHEMISTRY DEPARTMENT)


ProPath, a progressive, CAP accredited, high-volume pathology practice in 
Dallas, Texas is seeking an Histology Technician for its' Immunohistochemistry 
Lab.
Responsibilities include slide preparation (paraffin and frozen sections), IHC 
staining using our unique manual system, antibody titer preparation,
equipment maintenance, supply/reagent inventory maintenance, and QC/QA 
recording.

The ideal candidate will have a minimum of 4 years Histology experience with 
paraffin microtomy with a variety of different tissue types.
Working knowledge of IHC theory required, hands on IHC performance is desired.
We will train the ideal candidate to perform our manual IHC system.   HT (ASCP) 
strongly desired.

The hours for the position are 4:00 p.m. to 12:30 a.m. Monday through Friday.

ProPath utilizes leading technology and is a quality oriented pathology 
laboratory.
Benefits include medical, dental, Short and Long Term Disability insurance, a 
matched 401K plan and more!

Don't Follow the Leader!  Join the Leader!

To apply, please visit www.propath.com

EEO/AA-M/F/disability/protected veteran status

Accessibility Accommodations
If you require an accommodation to navigate or apply to our careers site, 
please send your request to accessibil...@propath.com.



Pat Patterson, HTL(ASCP)
Manager, Immunohistochemistry
ProPath - The Leader in Pathology Services
1355 River Bend Drive
Dallas, TX 75247

214-237-1700 x 2027
214-237-1730 fax

To learn more about ProPath, please visit 
http://www.ProPath.com



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