[Histonet] Re: [IHCRG] CD8 on mouse tissue

Wed Feb 20 10:23:14 CST 2013

Anne,
How long do you leave your tissue in the fixative? If you have a protocol you are willing to share it would be most appreciated.

Thanks!!
Pam

Sent from my iPhone

On Feb 20, 2013, at 9:32 AM, "Lewin, Anne" <anne.lewin <@t> bms.com<mailto:anne.lewin <@t> bms.com>> wrote:

We have used the zinc fixation method with some success, but you have to be VERY careful of the amount of time the tissue is in Zinc fix (NOT Zinc Formalin).  Also, make sure that your tissue processor has no trace of formalin in the lines or in the first alcohols during processing.
-Anne

From: ihcrg <@t> googlegroups.com<mailto:ihcrg <@t> googlegroups.com> [mailto:ihcrg <@t> googlegroups.com] On Behalf Of pruegg <@t> ihctech.net<mailto:pruegg <@t> ihctech.net>
Sent: Thursday, February 14, 2013 4:01 PM
To: ihcrg <@t> googlegroups.com<mailto:ihcrg <@t> googlegroups.com>; histonet <@t> lists.utsouthwestern.edu<mailto:histonet <@t> lists.utsouthwestern.edu>
Subject: FW: [IHCRG] CD8 on mouse tissue

Here is the reference for zinc salt fixed mouse tissue labeled for cd4/8 PE, it looks really promising.

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

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From: pruegg <@t> ihctech.net<mailto:pruegg <@t> ihctech.net> [mailto:pruegg <@t> ihctech.net]
Sent: Thursday, February 14, 2013 1:57 PM
To: pruegg <@t> ihctech.net<mailto:pruegg <@t> ihctech.net>
Subject: FW: [IHCRG] CD8 on mouse tissue

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

From: AJ Milici [mailto:amilici <@t> flagshipbio.com]
Sent: Thursday, February 14, 2013 11:56 AM
To: pruegg <@t> ihctech.net<mailto:pruegg <@t> ihctech.net>; Sherri Saturley
Subject: RE: [IHCRG] CD8 on mouse tissue

This will give you some info on my warped logic.
AJ

<image001.png>
A.J. Milici, Ph.D.
Director of Neurobiology and Inflammation
Flagship Biosciences
email: amilici <@t> flagshipbio.com<mailto:amilici <@t> flagshipbio.com>
Office: (203) 627-3369
www.flagshipbio.com<http://www.flagshipbio.com/>
This e-mail message, including any attachments, is for the sole use of the intended recipients and may contain privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by e-mail and destroy all copies of the original message. You may contact us at Flagship Biosciences LLC, 10955 Westmoor Dr, Suite 400, Westminster, CO 80021. Phone: 303-325-5894<tel:303-325-5894>

From: pruegg <@t> ihctech.net<mailto:pruegg <@t> ihctech.net> [mailto:pruegg <@t> ihctech.net]
Sent: Thursday, February 14, 2013 11:30 AM
To: AJ Milici; Sherri Saturley
Subject: RE: [IHCRG] CD8 on mouse tissue

The snap frozen in OCT tissue is fixed after sectioning in acetone/alcohol instead of formaldehyde, that is what I mean by non-aldehyde fixation.  CD4 and CD8 on murine tissue does not like formalin fixation and paraffin processing,  which is why the golden standard for those two is snap frozen murine tissue acetone/ethanol fixed.  I am looking into using zinc and/or PLP fixation for this.

Regards,
Patsy

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

From: AJ Milici [mailto:amilici <@t> flagshipbio.com]
Sent: Thursday, February 14, 2013 7:47 AM
To: pruegg <@t> ihctech.net<mailto:pruegg <@t> ihctech.net>; Sherri Saturley
Subject: RE: [IHCRG] CD8 on mouse tissue

Patsy:

I will start putting together my proposal and you should get it by Monday.  I have one question for you based upon what you wrote below – exactly what do you mean by non-aldehyde fixed frozen samples.  Do you mean fresh frozen sections that are fixed on the slide with cold MeOH or cold acetone?

Thanks,
AJ

<image001.png>
A.J. Milici, Ph.D.
Director of Neurobiology and Inflammation
Flagship Biosciences
email: amilici <@t> flagshipbio.com<mailto:amilici <@t> flagshipbio.com>
Office: (203) 627-3369
www.flagshipbio.com<http://www.flagshipbio.com/>
This e-mail message, including any attachments, is for the sole use of the intended recipients and may contain privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by e-mail and destroy all copies of the original message. You may contact us at Flagship Biosciences LLC, 10955 Westmoor Dr, Suite 400, Westminster, CO 80021. Phone: 303-325-5894<tel:303-325-5894>

From: pruegg <@t> ihctech.net<mailto:pruegg <@t> ihctech.net> [mailto:pruegg <@t> ihctech.net]
Sent: Wednesday, February 13, 2013 11:18 PM
To: AJ Milici; Sherri Saturley
Subject: RE: [IHCRG] CD8 on mouse tissue

We have done a great deal of CD8 localization in murine tissue but it has all been done on non aldehyde fixed frozen tissue, we have actually tried several antibodies on ffpe murine tissue for cd8 (the work we did for Roche/ John Woods) but did not have success.  I will inquire about zinc fixed samples and plp fixed samples for cd8 expression in murine tissues, I have heard of this but have not done it myself. If you have some references for zinc fixed murine tissues IHC stained for cd8 please forward those to us.

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

From: AJ Milici [mailto:amilici <@t> flagshipbio.com]
Sent: Wednesday, February 13, 2013 2:47 PM
To: pruegg <@t> ihctech.net<mailto:pruegg <@t> ihctech.net>; Sherri Saturley
Subject: RE: [IHCRG] CD8 on mouse tissue

I fully realize that CD8 localization in FFPE will not work.  However, there is some interesting data out there using a zinc fixative or PLP. While Frozen sections will work for the IHC they may be limiting in terms of the image analysis due to thicker sections and decreased morphological preservation.  I will lay out my data for you to poke holes in it.  So did you ever do much in the way of CD8 localization on murine tissue?

AJ

<image001.png>
A.J. Milici, Ph.D.
Director of Neurobiology and Inflammation
Flagship Biosciences
email: amilici <@t> flagshipbio.com<mailto:amilici <@t> flagshipbio.com>
Office: (203) 627-3369
www.flagshipbio.com<http://www.flagshipbio.com/>
This e-mail message, including any attachments, is for the sole use of the intended recipients and may contain privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by e-mail and destroy all copies of the original message. You may contact us at Flagship Biosciences LLC, 10955 Westmoor Dr, Suite 400, Westminster, CO 80021. Phone: 303-325-5894<tel:303-325-5894>

From: pruegg <@t> ihctech.net<mailto:pruegg <@t> ihctech.net> [mailto:pruegg <@t> ihctech.net]
Sent: Wednesday, February 13, 2013 3:57 PM
To: Sherri Saturley; AJ Milici
Subject: FW: [IHCRG] CD8 on mouse tissue

I am inquiring about advances in CD8 IHC on mouse tissue, here is one report, just as I thought, still.  AJ do you want to do this on formalin fixed paraffin embedded mouse tissue?  The standard for CD4 and CD8 still seems to be rat anti mouse ab on frozen samples not aldehyde fixed.

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

From: Brian Johnson [mailto:brianj18 <@t> u.washington.edu]
Sent: Wednesday, February 13, 2013 1:30 PM
To: pruegg <@t> ihctech.net<mailto:pruegg <@t> ihctech.net>
Subject: Re: [IHCRG] CD8 on mouse tissue

I just recently worked this up on Fresh Frozen vs. fixed, sucrose post fixed frozen and FFPE tissue.  Only worked on fresh frozen.  I use the BD marker that everyone swears by.  Same results for CD4.

I can give you more details if you are interested.

Brian

Brian Johnson
Program Manager
U.W. at South Lake Union, N310
HIC/Comparative Pathology Program
850 Republican St
Seattle, WA 98109-4725
Work Phone: 206-685-6517
Work E-mail: brianj18 <@t> uw.edu<mailto:brianj18 <@t> uw.edu>

www.uwhistologyandimaging.org<http://www.uwhistologyandimaging.org>

On Feb 13, 2013, at 12:22 PM, <pruegg <@t> ihctech.net<mailto:pruegg <@t> ihctech.net>> wrote:

Is this still being done on frozen sections not aldehyde fixed or has someone figured out an antibody or method of doing it on FFPE mouse tissue yet???

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

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