[IHCRG] RE: [Histonet] Mouse GranzymeB

Tue Jan 28 14:14:01 CST 2014

I agree with what Liz has said.  Usually when I encounter undesirable bg
staining especially if it is nuclear it most often points to over retrieval
of tissues not adequately fixed.  Besides using less harsh retrieval such as
low ph HIER, EIER, or no IER Loui Harkin is a big believer of prefixing
questionably fixed tissue sections after depar in formalin before HIER and
that has worked for me in the past as well.  I almost routinely use serum
free protein block (casein) and in difficult cases replace that with 20%
serum from the species the secondary is made in.  Dako anti rab Envision+ LP
and Leica's anti rab Power Vision LP are my go too reagents for using rab
antibodies on ffpe mouse tissue.  Leica Bonds detection system can be used
for this as well if u skip the post primary which is rabbit anti mouse and
just go direction to the goat anti rab labeled polymer detection.

Good luck,

Patsy

Patsy Ruegg, HT(ASCP)QIHC
Ruegg IHC Consulting
40864 E. Arkansas Ave
Bennett, CO 80102
H 303-644-4538
C 720-281-5406
pruegghm <@t> hotmail.com 

-----Original Message-----
From: ihcrg <@t> googlegroups.com [mailto:ihcrg <@t> googlegroups.com] On Behalf Of
Elizabeth Chlipala
Sent: Tuesday, January 28, 2014 12:26 PM
To: pruegg <@t> ihctech.net; 'Amos Brooks'; histonet <@t> lists.utsouthwestern.edu;
'ihcrg Group, (E-mail)'
Subject: RE: [IHCRG] RE: [Histonet] Mouse GranzymeB

Patsy and Amos

I can appreciate your comment but we have been using Dako's Envision+ Rabbit
on mouse tissue for years now and have never seen any cross reactivity to
mouse tissue, it's pretty much our go to detection system for rabbit
antibodies (both monoclonal and polyclonal) on mouse tissue.  We have
however seen cross reactivity to porcine with Dako's envision +mouse and
other companies anti-mouse polymer reagents.   My suggestion if you have not
already done this is to try to use some other retrieval methods such as pH6
or enzyme (proteinase K or pepsin or even no retrieval).  Tissue fixation is
important along with time an temp of retrieval - under fixation or over
retrieval or a combination of both can cause problems.  If we find we are
getting some background we will run some more intense blocking - such as the
following:

1. commercially available superblock - these can be competitive and may
potentially cause decreased sensitivity so you will need to watch out on
these 2.  Normal serum - we normally use a commercial serum free protein
block but on occasions we will use up to a 20% normal serum block for 30 to
60 minutes, if you want to continue to use the envision+ rabbit your choice
would be 20% normal goat serum 3. Saturated Casein - we'll make this up in
house

All are worth a try, good luck

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Premier Laboratory, LLC PO Box
18592 Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
liz <@t> premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-----Original Message-----
From: ihcrg <@t> googlegroups.com [mailto:ihcrg <@t> googlegroups.com] On Behalf Of
pruegg <@t> ihctech.net
Sent: Tuesday, January 28, 2014 12:11 PM
To: 'Amos Brooks'; histonet <@t> lists.utsouthwestern.edu; 'ihcrg Group,
(E-mail)'
Subject: [IHCRG] RE: [Histonet] Mouse GranzymeB

Amos u might have to get an anti rabbit link that has been mouse absorbed.
Dako's anti rab envision is made in a goat but it is not mouse absorbed.
Southern Biotec or Jackson Labs are really good places to get absorbed
links.  Still no reliable cd4 and cd8 for ffpe mouse tissue as far as I
know, I have tried many that claim they work but have not consistently
worked in my hands, we still do that with frozen mouse tissue not aldehyde
fixed.  Are u doing HIER with the EDTA ph9?  In my experience especially
with the higher ph u can over retrieve and cause undesirable nuclear
staining.

Patsy Ruegg, HT(ASCP)QIHC
Ruegg IHC Consulting
40864 E. Arkansas Ave
Bennett, CO 80102
H 303-644-4538
C 720-281-5406
pruegghm <@t> hotmail.com
rueggihcconsultingpr <@t> outlook.com

This email is confidential and intended solely for the use of the Person(s)
('the intended recipient') to whom it was addressed. Any views or opinions
presented are solely those of the author. It may contain information that is
privileged & confidential within the meaning of applicable law. Accordingly
any dissemination, distribution, copying, or other use of this message, or
any of its contents, by any person other than the intended recipient may
constitute a breach of civil or criminal law and is strictly prohibited. If
you are NOT the intended recipient please contact the sender and dispose of
this e-mail as soon as possible.

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Amos Brooks
Sent: Friday, January 24, 2014 10:09 AM
To: histonet <@t> lists.utsouthwestern.edu; ihcrg Group, (E-mail)
Subject: [Histonet] Mouse GranzymeB

Ok Musketeers,
     I am trying to detect cytotoxic T-Cells in formalin fixed paraffin
embedded mouse liver. GranzymeB should do the trick. I have a rabbit anti
mouse (and human and rat) GranzymeB from abCam (ab53097). The spleen that I
ran as a control with it works fine. Nice T-cells and no signifigant
background staining. The liver on the others hand has some lymphocyte
staining, presumably cytotoxic T-cells as expected,  but it looks like every
hepatocyte nuclei are also picking it up.  Grrr,  right?
     If this antibody had been raised in mouse,  I wouldn't be surprised.
Also,  if the spleen looked similar, I wouldn't be surprised. Do any of you
have any ideas ab pi ut what this might be? Alternatively, does anyone have
any ideas about another antibody that would work here?  CD4 or CD8 would be
great here,  but its FFPE mouse,  so sadly that's out (more grumbling).
    For the completionists out there,  I used EDTA pH9,  peroxide block and
detected the antibody (diluted 1:800) with Envision (rabbit) and DAB from
Dako.

Any suggestions would be vastly appreciated,

Happy Friday,
Amos
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

--
--
You received this message because you are subscribed to the Google Groups
"ihcrg" group. The IHC Resource Group is a standing committee within the
National Society for Histotechnology.

To post to this group, send email to ihcrg <@t> googlegroups.com To unsubscribe
from this group, send email to
ihcrg+unsubscribe <@t> googlegroups.com
For more options, visit this group at
http://groups.google.com/group/ihcrg?hl=en

To contact the National Society for Histotechnology, email: histo <@t> nsh.org or
call 443.535.4060.

---
You received this message because you are subscribed to the Google Groups
"ihcrg" group.
To unsubscribe from this group and stop receiving emails from it, send an
email to ihcrg+unsubscribe <@t> googlegroups.com.
For more options, visit https://groups.google.com/groups/opt_out.

--
--
You received this message because you are subscribed to the Google Groups
"ihcrg" group. The IHC Resource Group is a standing committee within the
National Society for Histotechnology.

To post to this group, send email to ihcrg <@t> googlegroups.com To unsubscribe
from this group, send email to
ihcrg+unsubscribe <@t> googlegroups.com
For more options, visit this group at
http://groups.google.com/group/ihcrg?hl=en

To contact the National Society for Histotechnology, email: histo <@t> nsh.org or
call 443.535.4060.

---
You received this message because you are subscribed to the Google Groups
"ihcrg" group.
To unsubscribe from this group and stop receiving emails from it, send an
email to ihcrg+unsubscribe <@t> googlegroups.com.
For more options, visit https://groups.google.com/groups/opt_out.