[Histonet] Immuno. confusion
Hi folks... I am going to try to revamp out Immuno. procedure and am looking at a couple of spec. sheets from our primary vendor. I am gettin conflicting information...so I am turning to you all for help. When doing immuno's that require either HIER or enzyme digestion, do you perform the retrieval first or do you do the peroxide step first?? One spec. sheets says retrieve then block endogenous peroxidase...the other says block endogenous peroxidase then do the HIER or enzyme digestion..which is correct?? One other question that I have is...what do you use to enhance your stain?? The enhancing solution that we were using has been discontinued. Thanks so much!! Valerie Hannen,MLT(ASCP), HTL,SU(FL) Histotechnologist Parrish Medical Center 951 N. Washington Avenue Titusville, Florida 32796 (321) 268-6111 ext. 7506 ** This email is intended solely for the use of the individual to whom it is addressed and may contain information that is privileged, confidential or otherwise exempt from disclosure under applicable law. If the reader of this email is not the intended recipient or the employee or agent responsible for delivering the message to the intended recipient, you are hereby notified that any dissemination, distribution, or copying of this communication is strictly prohibited. If you have received this communication in error, please immediately delete this message. Thank you ** ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Immuno. confusion
If you fix with formaldehyde, HIER will eliminate the cross link and that step is necessary BEFORE you can apply the peroxide. The enzyme (peroxidase) is also a protein and has to be open to receive the H202. The same if you want to apply enzymatic digestion, HIER has to be done first. HIER will remove the formaldehyde cross linking and this step is required before any other in the IHC protocol. René J. --- On Wed, 8/3/11, Hannen, Valerie valerie.han...@parrishmed.com wrote: From: Hannen, Valerie valerie.han...@parrishmed.com Subject: [Histonet] Immuno. confusion To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Date: Wednesday, August 3, 2011, 11:57 AM Hi folks... I am going to try to revamp out Immuno. procedure and am looking at a couple of spec. sheets from our primary vendor. I am gettin conflicting information...so I am turning to you all for help. When doing immuno's that require either HIER or enzyme digestion, do you perform the retrieval first or do you do the peroxide step first?? One spec. sheets says retrieve then block endogenous peroxidase...the other says block endogenous peroxidase then do the HIER or enzyme digestion..which is correct?? One other question that I have is...what do you use to enhance your stain?? The enhancing solution that we were using has been discontinued. Thanks so much!! Valerie Hannen,MLT(ASCP), HTL,SU(FL) Histotechnologist Parrish Medical Center 951 N. Washington Avenue Titusville, Florida 32796 (321) 268-6111 ext. 7506 ** This email is intended solely for the use of the individual to whom it is addressed and may contain information that is privileged, confidential or otherwise exempt from disclosure under applicable law. If the reader of this email is not the intended recipient or the employee or agent responsible for delivering the message to the intended recipient, you are hereby notified that any dissemination, distribution, or copying of this communication is strictly prohibited. If you have received this communication in error, please immediately delete this message. Thank you ** ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Immuno. confusion
Hi Valerie, The peroxidase blocking step is to prevent endogenous peroxidase activity that will cause a false positive stemming from your application of DAB (the substrate for your DAB solution is hydrogen peroxide). You can essentially put this blocking step at any point in your stain (following deparaffinization) and before your application of DAB. I have run this step at varying points in my procedures and they all work fine. Some antibodies prefer you do this step before primary application and others prior to DAB, but for most stains, it doesn't matter too much. As for an enhancer, you can make a copper sulfate solution (5mg/mL in buffer). Or you can try DAB Enhancer or DAB Sparkle from Biocare. Good luck, Ashley Troutman BS, HT(ASCP) QIHC Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 From: Hannen, Valerie valerie.han...@parrishmed.com Subject: [Histonet] Immuno. confusion To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Date: Wednesday, August 3, 2011, 11:57 AM Hi folks... I am going to try to revamp out Immuno. procedure and am looking at a couple of spec. sheets from our primary vendor. I am gettin conflicting information...so I am turning to you all for help. When doing immuno's that require either HIER or enzyme digestion, do you perform the retrieval first or do you do the peroxide step first?? One spec. sheets says retrieve then block endogenous peroxidase...the other says block endogenous peroxidase then do the HIER or enzyme digestion..which is correct?? One other question that I have is...what do you use to enhance your stain?? The enhancing solution that we were using has been discontinued. Thanks so much!! Valerie Hannen,MLT(ASCP), HTL,SU(FL) Histotechnologist Parrish Medical Center 951 N. Washington Avenue Titusville, Florida 32796 (321) 268-6111 ext. 7506 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Immuno. confusion
I have seen this done many ways, some people have h202 at th= e end of
their depara setup if doing that off line. For the Leica Bon= d
instrument we were advised to do h202 block just before the DAB and i
hav= e noticed that it works best that way for their system. I for
years h= ave always used h202 after the primary ab as some abs are
affected by it so= I just don't take the chance of not knowing which
may be. There was = one ab, cd4 I believe that for whatever reason
needed to be h202 blocked be= fore AR and the AB, and it really didn't
work if you did not do the block b= efore everything. Just goes to
show that each ab has to be handled sp= ecifically.
A little aside here and techni= cally it makes no sense but we were
having trouble with the Leica ap/red ki= t preciptating and was
advised by Jim Burchette not Leica to use H202 = just before the red
substrate chromogen and that has really improved the ap= /red staining
for us which we use a lot on derm samples. I know = endogenous alk
phos uses levamisol as a block and not h202 but for some rea= son this
works so I don't argue with it.
R= egards,
Patsy
Original Message Subject: [Histonet] Immuno.
confusion
From: Hannen, Valerie valerie.han...@parrishmed.com= /a
Date: Wed, August 03, 2011 8:57 am
To: [1]histonet@lists.utsouthwestern.edu
= lt;[2]histo...@lists.utso= uthwestern.edu
Hi folks...
I am going to try to revam= p out Immuno. procedure and am looking at
a couple of spec. sheets from our= primary vendor. I am gettin
conflicting information...so I am turning to y= ou all for help.
When doing immuno's that require either HIER or enz= yme digestion, do
you perform the retrieval first or do you do the peroxid= e step
first??
One spec. sheets says retrieve then block endogenous per
oxidase...the other says block endogenous peroxidase then do the HIER
or en= zyme digestion..which is correct??
One other question that I have is= ...what do you use to enhance your
stain?? The enhancing solution that we w= ere using has been
discontinued.
Thanks so much!!
Valerie Han= nen,MLT(ASCP), HTL,SU(FL)
Histotechnologist
Parrish Medical Center
951 N. Washington Avenue
Titusville, Florida 32796
(321) 268-6111 ex= t. 7506
= **
This email is intended solely for the use of the individual = to
whom it is addressed and may contain information that is
privilege= d, confidential or otherwise exempt from disclosure
under applicable law= . If the reader of this email is not the
intended recipient or the emplo= yee or agent responsible for
delivering the message to the intended reci= pient, you are
hereby notified that any dissemination, distribution, or= BRcopying
of this communication is strictly prohibited. If you
have rec= eived this communication in error, please immediately
delete this messag= e. Thank you
**=
___
Histonet mailing= list
[3]Histonet@list= s.utsouthwestern.edu
[4]http://lists.utsouthwestern.edu/mailman/listinfo/his= tonet
References
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[Histonet] Immuno. confusion
Hi, Well... almost any point before DAB. You wouldn't want to put it in after the HRP conjugated secondary because that would quench the HRP signal you are actually looking for leaving you nothing for the DAB to precipitate on. On the other hand you certainly do not *need* to do it after the HIER as it will work just fine before it or even after the primary antibody (assuming it isn't HRP conjugated). Actually we have had the best results on CD4 if the H2O2 is done prior to the HIER. The H2O2 in this case actually affects the epitope if you do it after HIER. If it does that with CD4, I assume it is likely to do so with other finicky epitopes. Have a nice day, Amos On Wed, Aug 3, 2011 at 1:00 PM, histonet-requ...@lists.utsouthwestern.eduwrote: Message: 7 Date: Wed, 3 Aug 2011 11:49:40 -0500 From: Troutman, Kenneth A ashley.trout...@vanderbilt.edu Subject: [Histonet] Immuno. confusion To: Histonet@lists.utsouthwestern.edu Histonet@lists.utsouthwestern.edu Message-ID: 7b310892042da74cb3590053f424cfe6143ee30...@its-hcwnem06.ds.vanderbilt.edu Content-Type: text/plain; charset=us-ascii Hi Valerie, The peroxidase blocking step is to prevent endogenous peroxidase activity that will cause a false positive stemming from your application of DAB (the substrate for your DAB solution is hydrogen peroxide). You can essentially put this blocking step at any point in your stain (following deparaffinization) and before your application of DAB. I have run this step at varying points in my procedures and they all work fine. Some antibodies prefer you do this step before primary application and others prior to DAB, but for most stains, it doesn't matter too much. As for an enhancer, you can make a copper sulfate solution (5mg/mL in buffer). Or you can try DAB Enhancer or DAB Sparkle from Biocare. Good luck, Ashley Troutman BS, HT(ASCP) QIHC Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
