[Histonet] Gallocyanine

[Eva Permaul via Histonet]

Good morning out there,
We just had a request for Gallocyanine staining. Does anyone do this? Can
you share your protocol? Where do you buy your reagents?
Thanks in advance for any help in the right direction,
Eva Permaul
Georgetown University
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

[Histonet] Can't get the calculation right today

[Eva Permaul via Histonet]

Please, please and please help me. I am fighting a sinus infection and I
can not think to save my life.
I have to make a buffer solution:

10mM Tris-HCL, 5mM CaCl2, 50% glycerol (v/v), pH7

I have tris base and calcium chloride powder.

Can someone please help me by writing it out for me?
I am so stuck.

Thank you,
Eva
(the one whose head is about to explode)
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Re: [Histonet] Human nuclear antibody MAB1281

[Eva Permaul via Histonet]

Liz,
Any chance you could find out what protocol was used by the ones that have
used it? or help me find some of the literature references? Everywhere I
look I just see people who have not gotten it to work asking for help.
Thanks,
Eva

On Thu, Feb 25, 2016 at 10:39 AM, Elizabeth Chlipala 
wrote:

> Eva
>
> We have worked with various antibodies in order to detect human cells in a
> rodent background.  I know that this antibody has been referenced in the
> literature and I also have known of other individuals who have had success
> with it, but in our hands we have never been able to make it work on FFPE
> tissues but I believe it works in frozen sections.  We have opted to try
> some different antibodies.
>
> NuMA -we researched this protein and determined that it may work -here is
> a snippet of what we found from a quick look into the Human Protein Atlas
> entry for NUMA1. Though it is functionally involved in the structural
> rearrangement of DNA during mitosis and apoptosis, it is a constitutively
> expressed structural protein. From the images on HPA (
> http://www.proteinatlas.org/ENSG0137497-NUMA1/tissue/primary+data)
> it looks like it is almost universally expressed, with the one exception of
> hepatocytes. The vast majority of neurons, which are overwhelmingly
> post-mitotic, show fairly strong expression, so that's encouraging.   We
> initially looked at the cell signaling antibody (our target was human cells
> in a rat background) but found that it does cross react with rat, its fine
> with mouse but this antibody is our hands was found to be sensitive to time
> in fixation and retrieval time and temp.  We opted to try another source
> for this antibody - abcam.   They had several different antibodies for that
> target.  We work with their antibodies a lot and they normally do not list
> the sequence of the antigen so we could not check the sequence homology to
> other species ourselves but you can call them and they will provide that
> information.  Here is what they provided to us -   "We have not tested
> ab86129 for cross reactivity with rat and we have not received any
> researcher feedback for using the antibody with this species. The immunogen
> sequence has 56% identity with the rat protein, so it is unlikely that it
> will cross react. However we have not confirmed this experimentally and we
> do not know if it would show non-specific staining with rat. I do think
> ab86129 would be the best antibody to try since it has the lowest homology
> with the rat protein. The other antibodies that have not been tested with
> rat have 69% (ab55767) or 88% (ab84680) homology."  Ab86129 worked very
> nicely in our hands.
>
> HLA-1 this will also work nicely but the protein is expressed in the
> cytoplasm and expression levels can vary but it does work nicely in some
> instances.
>
> So for us its dependent upon the project as to which antibody we will use
> but the NuMA is expressed in the nuclei so it will work quite nicely for
> dual staining of another protein that is expressed in the cytoplasm or cell
> membrane.
>
> 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
> l...@premierlab.com
> www.premierlab.com
>
> Ship to Address:
>
> Premier Laboratory, LLC
> 1567 Skyway Drive, Unit E
> Longmont, CO 80504
>
> -Original Message-
> From: Eva Permaul via Histonet [mailto:histonet@lists.utsouthwestern.edu]
> Sent: Thursday, February 25, 2016 7:07 AM
> To: histonet
> Subject: [Histonet] Human nuclear antibody MAB1281
>
> Good morning,
> We recently bought MAB1281 with the hope of being able to determine if the
> cells in a mouse model was human or mouse.
> We want to use it to stain FFPE tissues. I have tried it with Citrate per
> the companies recommendation. I ran it on a human tonsil but saw no
> staining at all.
> Is there anyone who has been able to get this antibody to work? And if so
> would you please share your protocol?
> Thank you,
> Eva
> ___
> 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] Human nuclear antibody MAB1281

[Eva Permaul via Histonet]

Good morning,
We recently bought MAB1281 with the hope of being able to determine if the
cells in a mouse model was human or mouse.
We want to use it to stain FFPE tissues. I have tried it with Citrate per
the companies recommendation. I ran it on a human tonsil but saw no
staining at all.
Is there anyone who has been able to get this antibody to work? And if so
would you please share your protocol?
Thank you,
Eva
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

[Histonet] Ki67 antibody detecting mouse

[Eva Permaul]

Good morning,
We used to have a ki67 antibody that detected mouse that worked really
well. It was made in rat. It is however now really weak. We have received
two different lots and it is fading fast. Could anyone recommend one that
works well for IHC-P on mouse tissues? We would prefer if it was not made
in mouse. Rabbit, Rat or Goat would be the best. Also if it has been
published it would be great to know.
Thank you,
Eva
Georgetown University
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

[Histonet] Mount-Quick for tissue transfer?

[Eva Permaul]

Good morning Histonetters,
I was wondering if anyone has used Mount-Quick to transfer tissue from
uncharged slides to charged slides. We had a submitter give us slides for
staining on uncharged slides and the tissue comes off the slide during
antigen retrieval. I remember reading about this product but have never
used it. Is it hard to do? Are the slides staining well afterwards?
Thank you,
Eva
Georgetown University
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

[Histonet] HA-tag antibody

[Eva Permaul]

Hello,
I am looking for an HA-tag antibody to detect a protein labeled with HA in
formalin fixed paraffin embedded tissues. Could anyone suggest a published
antibody? The tissue of interest is mouse so I would prefer an antibody
made in another species.
I have found several antibodies but can't find them published.
Thank you,
Eva
Georgetown University
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Re: [Histonet] antibody diluent

[Eva Permaul]

We usually just use 1xTris buffered saline with 0.05% Tween.

Eva


On Tue, Apr 9, 2013 at 3:34 PM, Geoff  wrote:

> You could make your own. It is usually just a buffer with some normal
> serum and Triton or Tween added.
>
> Geoff
>
>
> On 4/9/2013 2:14 PM, Perry, Margaret wrote:
>
>> I am looking for a less expensive antibody diluent and would like some
>> opinions on which companies are best .  Are there differences between
>> diluents when it comes to background staining?
>>
>> Margaret Perry HT(ASCP)
>> Veterinary & Biomedical Sciences Department
>> North Campus Drive Box 2175
>> South Dakota State University
>> Brookings SD 57007
>> 605-688-5638
>>
>> __**_
>> Histonet mailing list
>> Histonet@lists.utsouthwestern.**edu 
>> http://lists.utsouthwestern.**edu/mailman/listinfo/histonet
>>
>
>
> --
> --
> 
> Geoff McAuliffe, Ph.D.
> Neuroscience and Cell Biology
> Robert Wood Johnson Medical School
> 675 Hoes Lane, Piscataway, NJ 08854
> voice: (732)-235-4583; fax: -4029
> mcaul...@umdnj.edu
> 
>
>
>
>
> __**_
> 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] Re: Peptide needed for peptide competition

[Eva Permaul]

Does anyone have any suggestions?


On Wed, Oct 24, 2012 at 8:26 AM, Eva Permaul  wrote:

> Good morning,
> I need to do a peptide competition for one of the antibodies we are using
> for IHC on FFPE. The company does not offer the peptide but the datasheet
> does have the sequence. Who do you use to generate peptides? Can you
> recommend any particular company?
> Thank you in advance for all of your valuable advice,
> Eva Permaul
> Georgetown University
>
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

[Histonet] Peptide needed for peptide competition

[Eva Permaul]

Good morning,
I need to do a peptide competition for one of the antibodies we are using
for IHC on FFPE. The company does not offer the peptide but the datasheet
does have the sequence. Who do you use to generate peptides? Can you
recommend any particular company?
Thank you in advance for all of your valuable advice,
Eva Permaul
Georgetown University
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Re: [Histonet] Albumin and Epcam to stain mouse cell pellets

[Eva Permaul]

Thank you Anatoli.

Let me add some more info to my question. The samples are FFPE.
Thanks,
Eva

On Tue, Sep 25, 2012 at 2:43 PM, Anatoli Gleiberman <
agleiber...@cbiolabs.com> wrote:

> Eva
> For mouse EpCAM I have used routinely during past 15 years G8.8 rat
> monoclonal antibody from Developmental Studies Hybridoma Bank. Concentrated
> supernatant (1:200 dilution for IF) works very well on 4% paraformaldehyde
> fixed frozen samples (or on sections from fresh-frozen samples post-fixed
> with formaldehyde). Epitope is very sensitive to alcohol, so even fixation
> with NBF instead of formaldehyde diminishes staining dramatically. I was
> told that this antibody works on formalin-fixed paraffin sections after
> HIER retrieval, but never got results comparable to frozen sections. Don't
> have any recommendations regarding albumin, I used home-made antibodies for
> that purpose many years ago. In general, paraformaldehyde fixation and
> frozen sections worked satisfactory. Classical approach for albumin and
> alpha-fetoprotein staining was to fix in Saint-Mary fixative (1% acetic
> acid in absolute ethanol) following routine paraffin embedding.
> Nevertheless, short fixation with NBF (4h at room temp, following PBS wash)
> and paraffin embedding works as well. Best results regarding liver secreted
> proteins (as well as all membrane and cytoskeletal markers) could be
> achieved on cryo-sections after fixation by perfusion with 4%
> paraformaldehyde in PBS.
>
> Anatoli Gleiberman, PhD
> Director of Histopathology
> Cleveland Biolabs, Inc
> 73 High Street
> Buffalo, NY 14203
> phone:716-849-6810 ext.354
> fax:716-849-6817
> e-mail: agleiber...@cbiolabs.com
>
> -Original Message-
> From: histonet-boun...@lists.utsouthwestern.edu [mailto:
> histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Eva Permaul
> Sent: Tuesday, September 25, 2012 1:21 PM
> To: histonet
> Subject: [Histonet] Albumin and Epcam to stain mouse cell pellets
>
> Good afternoon,
> Has anyone used Albumin and Epcam antibodies to stain mouse samples? If
> you have would you please share the antibody information and conditions
> with me?
> Thank you,
> Eva Permaul
> Georgetown University
> ___
> 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] Albumin and Epcam to stain mouse cell pellets

[Eva Permaul]

Good afternoon,
Has anyone used Albumin and Epcam antibodies to stain mouse samples? If you
have would you please share the antibody information and conditions with me?
Thank you,
Eva Permaul
Georgetown University
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

[Histonet] Pronase antigen retrieval

[Eva Permaul]

Hello out there in Histoland,
I have to do some bulk runs with Pronase antigen retrieval. Up until now
the number of slides have been so few that I have been able to do the
antigen retrieval by dropping pronase on each slide and incubating them in
a humidified chamber at 37C for 20min. I now have so many slides that my
manager has suggested that I make up large amounts of the Pronase (250ml)
and submerge the slides. She suggested placing the container in the oven
earlier to allow the Pronase to get to 37C before I add the slides. How
long is the Pronase ok to be keep at 37C before I use it? How long in
advance can I place it in the 37C before it looses its ability to be used
as an antigen retrieval?
Thanks for all your help,
Eva Permaul
HTSR
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Re: [Histonet] CD20 for Mouse Tissue

[Eva Permaul]

Hi Liz,
We just used CD20 cat.no. 14-0201 from eBioscience on mouse livers and
spleen. It worked great. Email me separate if you would like to send you
the protocol.
Eva

Eva Permaul
Georgetown University

On Wed, Aug 22, 2012 at 2:48 PM, Elizabeth Cameron <
elizabeth.came...@jax.org> wrote:

> Does anyone know of a CD20 antibody that works well on paraffin embedded
> mouse tissue?
> Thanks!
> -Liz
>
>
> The information in this email, including attachments, may be confidential
> and is intended solely for the addressee(s). If you believe you received
> this email by mistake, please notify the sender by return email as soon as
> possible.
> ___
> 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

Re: [Histonet] Ki67 antibody to stain Mouse tissue

[Eva Permaul]

I just found a nice publication with the Rm-9106 on mouse intestine. Have
provided it to my manager. Thank you everyone.
Eva

On Wed, Aug 8, 2012 at 11:38 AM, Eva Permaul  wrote:

> Just to clarify. Are you using it on mouse tissues? I am only asking
> because the Thermo Scientific/Labvision/Neomarkers data sheet says it has
> only been verified on Human tissues.
> Secondly had anyone used it on mouse gut. Does it stain in the bottom of
> the crypts or at the top? I am asking because I have also tried a different
> antibody from Leica but got staining at the top of the crypts not at the
> bottom.
>
> Thank you to everyone for all of your valuable suggestions and advice,
> Eva Permaul
> Georgetown University
>
>
> On Tue, Aug 7, 2012 at 2:21 AM, Bruijntjes, J.P. (Joost) <
> joost.bruijnt...@tno.triskelion.nl> wrote:
>
>> Hi Eva
>>
>> I've used an antibody from Labvision /Neomarkers. It is a rabbit
>> monoclonal; catalog number: RM-9106.
>>
>> Joost
>>
>> -Oorspronkelijk bericht-
>> Van: histonet-boun...@lists.utsouthwestern.edu [mailto:
>> histonet-boun...@lists.utsouthwestern.edu] Namens Eva Permaul
>> Verzonden: maandag 6 augustus 2012 20:44
>> Aan: histonet
>> Onderwerp: [Histonet] Ki67 antibody to stain Mouse tissue
>>
>> Hello,
>> I am looking for a Ki67 to stain mouse tissues. We have been using Dako
>> M2749 but just found out it has been discontinued. Could anyone suggest a
>> good alternative?
>> Thank you all for any help,
>> Eva Permaul
>> Georgetown University
>> ___
>> 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

Re: [Histonet] Ki67 antibody to stain Mouse tissue

[Eva Permaul]

Just to clarify. Are you using it on mouse tissues? I am only asking
because the Thermo Scientific/Labvision/Neomarkers data sheet says it has
only been verified on Human tissues.
Secondly had anyone used it on mouse gut. Does it stain in the bottom of
the crypts or at the top? I am asking because I have also tried a different
antibody from Leica but got staining at the top of the crypts not at the
bottom.

Thank you to everyone for all of your valuable suggestions and advice,
Eva Permaul
Georgetown University

On Tue, Aug 7, 2012 at 2:21 AM, Bruijntjes, J.P. (Joost) <
joost.bruijnt...@tno.triskelion.nl> wrote:

> Hi Eva
>
> I've used an antibody from Labvision /Neomarkers. It is a rabbit
> monoclonal; catalog number: RM-9106.
>
> Joost
>
> -Oorspronkelijk bericht-
> Van: histonet-boun...@lists.utsouthwestern.edu [mailto:
> histonet-boun...@lists.utsouthwestern.edu] Namens Eva Permaul
> Verzonden: maandag 6 augustus 2012 20:44
> Aan: histonet
> Onderwerp: [Histonet] Ki67 antibody to stain Mouse tissue
>
> Hello,
> I am looking for a Ki67 to stain mouse tissues. We have been using Dako
> M2749 but just found out it has been discontinued. Could anyone suggest a
> good alternative?
> Thank you all for any help,
> Eva Permaul
> Georgetown University
> ___
> 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

Re: [Histonet] Ki67 antibody to stain Mouse tissue

[Eva Permaul]

We are staining straight mouse tissue.

On Mon, Aug 6, 2012 at 2:46 PM, Sarah Dysart  wrote:

> Are you staining human xenografts in mice or straight out mouse tissue?  I
> get all mine from abcam.  I am currently staining xenografts so I am using
> a human specific one, but I think it might be good for mouse too?
>
> Sarah Goebel-Dysart, BA, HT(ASCP), QIHC (ASCP)
> Histotechnologist
> Mirna Therapeutics
> 2150 Woodward Street
> Suite 100
> Austin, Texas  78744
> (512)901-0900 ext. 6912
>
>
> -Original Message-
> From: histonet-boun...@lists.utsouthwestern.edu [mailto:
> histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Eva Permaul
> Sent: Monday, August 06, 2012 1:44 PM
> To: histonet
> Subject: [Histonet] Ki67 antibody to stain Mouse tissue
>
> Hello,
> I am looking for a Ki67 to stain mouse tissues. We have been using Dako
> M2749 but just found out it has been discontinued. Could anyone suggest a
> good alternative?
> Thank you all for any help,
> Eva Permaul
> Georgetown University
> ___
> 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] Ki67 antibody to stain Mouse tissue

[Eva Permaul]

Hello,
I am looking for a Ki67 to stain mouse tissues. We have been using Dako
M2749 but just found out it has been discontinued. Could anyone suggest a
good alternative?
Thank you all for any help,
Eva Permaul
Georgetown University
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Re: [Histonet] RE: Secondary antibody causing nuclear staining

[Eva Permaul]

I do see positive nuclei in the NC. That is what I am asking about. I know
I could switch methods but my question is also why if it is happening is it
not as strong all the time? Why are the cells very light one day and dark
the next? What is causing them to stain? Just curious is all.
Eva

On Wed, Jul 25, 2012 at 10:04 AM, Reynolds,Donna M
wrote:

>
> If you are running a negative control (no primary)with your ABC staining
> wouldn't you see the same nuclear labeling in the NC ? Thus alerting you to
> false staining and indicating that you should try a HRP conjugated
> secondary or use a polymer system.
> Good discussion thank Tony.
> Donna Reynolds HT(ASCP) Chief Histologist IHC Lab
>
> -Original Message-
> > I understand the point about the biotin and I should have said that
> > when using the ABC method we have taken to always using an
> > avidin/biotin blocking kit. We are using biotinylated secondary
> > antibodies from Vector. I have seen the same problem occur in our
> > anti-mouse, anti-rabbit and anti-goat. In my last run I had stomach
> > fundus as well as skin melanoma, both had pos.nuclei in the negative
> > (no primary). In another run I had colon ca and breast ca, the breast
> > ca had fewer pos. nuclei than the colon ca but they were still there.
> > Some days the positive nuclei are stronger in a sample that was just
> > weakly positive before. Just want to understand what it is and what
> effects it.
> > Thank you all for your ideas.
> > Eva Permaul
> > Georgetown University
> >
> > On Mon, Jul 23, 2012 at 7:16 PM, Tony Henwood (SCHN) <
> > tony.henw...@health.nsw.gov.au> wrote:
> >
> >> I should have added that this was from the workshop notes on a
> >> Hypotheticals Workshop I ran last year at our Australian National
> Meeting.
> >>
> >> 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 Tony Henwood
> >> (SCHN)
> >> Sent: Tuesday, 24 July 2012 9:00 AM
> >> To: 'Eva Permaul'; histonet@lists.utsouthwestern.edu
> >> Subject: RE: [Histonet] Secondary antibody causing nuclear staining?
> >>
> >> It is possible that this is due to "Biotin nuclei" where excess
> >> biotin is found in the nuclei of some cells, see below:
> >>
> >> Optically clear nuclei have been reported in endometrial epithelium
> >> associated with first and second trimester abortions (Sickel & di
> >> Sant'Agnese 1994). Optically clear nuclei have also been found in
> >> different types of tissues of diverse organs such as ovary, thyroid
> >> and lung (Nakatani et al 1994, Mount & Cooper 2001). The optically
> >> clear nuclei contain excess biotin.
> >>
> >> Endogenous biotin immunoreactivity is generally not visualized in
> >> formalin fixed, paraffin-embedded tissues unless a heat-induced
> >> antigen retrieval step has been introduced (Mount & Cooper 2001).
> >>
> >> In this placental section, optically clear nuclei (containing biotin)
> >> bind to the streptavidin of the ABC technique giving a reaction
> >> similar to that seen with CMV containing cells. If a polymer method
> >> (or even the original Sternberger's PAP method) is used then this
> >> anomalous staining will disappear, thus allowing confident
> demonstration of CMV infected nuclei.
> >>
> >> The false-positive staining pattern caused by endogenous biotin can
> >> be cytoplasmic or nuclear. A report of positive immunoreactivity of
> >> hepatocellular carcinomas for inhibin was later determined to be a
> >> false-positive finding due to cytoplasmic endogenous biotin. Steroid
> >> cell tumours of the ovary were found to demonstrate endogenous biotin
> >> cytoplasmic staining in 36% of cases. Immunoreactivity for
> >> anti-Herpes virus immunohistochemical staining in a series of
> >> endometria was also later determined to be a false-positive result
> >> due to biotin. The prominent intranuclear inclusions, resembling
> >> herpes virus cytopathic effect, were caused

Re: [Histonet] Secondary antibody causing nuclear staining?

[Eva Permaul]

Yes. The strength of the stained nuclei in the no primary slides are
stronger on some days than others.


On Tue, Jul 24, 2012 at 8:18 AM, Kim Donadio wrote:

> Are you getting false positives and variations on the same control tissue
> for different days ?
>
> Sent from my iPhone
>
> On Jul 24, 2012, at 8:13 AM, Eva Permaul  wrote:
>
> > I understand the point about the biotin and I should have said that when
> > using the ABC method we have taken to always using an avidin/biotin
> > blocking kit. We are using biotinylated secondary antibodies from
> Vector. I
> > have seen the same problem occur in our anti-mouse, anti-rabbit and
> > anti-goat. In my last run I had stomach fundus as well as skin melanoma,
> > both had pos.nuclei in the negative (no primary). In another run I had
> > colon ca and breast ca, the breast ca had fewer pos. nuclei than the
> colon
> > ca but they were still there. Some days the positive nuclei are stronger
> in
> > a sample that was just weakly positive before. Just want to understand
> what
> > it is and what effects it.
> > Thank you all for your ideas.
> > Eva Permaul
> > Georgetown University
> >
> > On Mon, Jul 23, 2012 at 7:16 PM, Tony Henwood (SCHN) <
> > tony.henw...@health.nsw.gov.au> wrote:
> >
> >> I should have added that this was from the workshop notes on a
> >> Hypotheticals Workshop I ran last year at our Australian National
> Meeting.
> >>
> >> 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 Tony Henwood
> >> (SCHN)
> >> Sent: Tuesday, 24 July 2012 9:00 AM
> >> To: 'Eva Permaul'; histonet@lists.utsouthwestern.edu
> >> Subject: RE: [Histonet] Secondary antibody causing nuclear staining?
> >>
> >> It is possible that this is due to "Biotin nuclei" where excess biotin
> is
> >> found in the nuclei of some cells, see below:
> >>
> >> Optically clear nuclei have been reported in endometrial epithelium
> >> associated with first and second trimester abortions (Sickel & di
> >> Sant'Agnese 1994). Optically clear nuclei have also been found in
> different
> >> types of tissues of diverse organs such as ovary, thyroid and lung
> >> (Nakatani et al 1994, Mount & Cooper 2001). The optically clear nuclei
> >> contain excess biotin.
> >>
> >> Endogenous biotin immunoreactivity is generally not visualized in
> formalin
> >> fixed, paraffin-embedded tissues unless a heat-induced antigen retrieval
> >> step has been introduced (Mount & Cooper 2001).
> >>
> >> In this placental section, optically clear nuclei (containing biotin)
> bind
> >> to the streptavidin of the ABC technique giving a reaction similar to
> that
> >> seen with CMV containing cells. If a polymer method (or even the
> original
> >> Sternberger's PAP method) is used then this anomalous staining will
> >> disappear, thus allowing confident demonstration of CMV infected nuclei.
> >>
> >> The false-positive staining pattern caused by endogenous biotin can be
> >> cytoplasmic or nuclear. A report of positive immunoreactivity of
> >> hepatocellular carcinomas for inhibin was later determined to be a
> >> false-positive finding due to cytoplasmic endogenous biotin. Steroid
> cell
> >> tumours of the ovary were found to demonstrate endogenous biotin
> >> cytoplasmic staining in 36% of cases. Immunoreactivity for anti-Herpes
> >> virus immunohistochemical staining in a series of endometria was also
> later
> >> determined to be a false-positive result due to biotin. The prominent
> >> intranuclear inclusions, resembling herpes virus cytopathic effect, were
> >> caused by intranuclear biotin and not viral particles. Similar false
> >> positive staining for CMV in products of conception has also been
> reported
> >> (Mount & Cooper 2001).
> >>
> >> False-positive staining can be cytoplasmic or nuclear. When cytoplasmic,
> >> the appearance of the false signal is that of 

Re: [Histonet] Non-Histo. question

[Eva Permaul]

Hi Sarah,
I had a similar situation. If you go into your profile on the Histonet site
you can click to not receive any of the Histonet messages while you are
away. Then when you get back and want the messages again you just change it
back.
Eva

On Tue, Jul 24, 2012 at 2:35 PM, Sarah Dysart  wrote:

> So, I go on maternity leave in 52 (I hope...) days...while out I will have
> one of those automatic messages that says I'm out...Since I get say 20ish
> emails a day from histonet I would assume that 20ish of these replys will
> go out to everyone, and I don't want to drive people bonkers.
>
> Maybe the moderator could answer...what do you want me to do?  Cancel and
> rejoin when I get back, or is this just something that gets filtered out?
> Thanks
>
> Sarah Goebel-Dysart, BA, HT(ASCP), QIHC (ASCP)
> Histotechnologist
> Mirna Therapeutics
> 2150 Woodward Street
> Suite 100
> Austin, Texas  78744
> (512)901-0900 ext. 6912
>
> ___
> 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

Re: [Histonet] Cyclin D1 IHC

[Eva Permaul]

We have used Neomarkers/Labvision cat.no. RM-9104 on mouse salivary glands
and mammary glands.

On Tue, Jul 24, 2012 at 11:16 AM, Suresch, Donna L.  wrote:

> Hello Histonetters,
> Has anyone done IHC using Cyclin D1 antibody?  What vendor was used?  What
> control tissue was used?
> Thank you.
> Donna Suresch - Merck & Co.
>
> Donna L. Suresch
> Imaging Research Scientist
> Merck Research Laboratories
> Department of Imaging - West Point Campus
> Mail Stop:  WP44KOffice: WP44-H129
> 770 Sumneytown Pike
> PO Box 4
> West Point, PA  19486-0004
> Phone:  215-652-7349
> Fax:  215-993-6803
>
>
> Notice:  This e-mail message, together with any attachments, contains
> information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station,
> New Jersey, USA 08889), and/or its affiliates Direct contact information
> for affiliates is available at
> http://www.merck.com/contact/contacts.html) that may be confidential,
> proprietary copyrighted and/or legally privileged. It is intended solely
> for the use of the individual or entity named on this message. If you are
> not the intended recipient, and have received this message in error,
> please notify us immediately by reply e-mail and then delete it from
> your system.
>
> ___
> 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

Re: [Histonet] Secondary antibody causing nuclear staining?

[Eva Permaul]

These samples were all human but I have seen it in Mouse mammary gland as
well but those nuclei were lighter.
Eva

On Tue, Jul 24, 2012 at 8:51 AM, James Burchette Jr. <
james.burche...@duke.edu> wrote:

> Thanks Eva. I don't know why you are having the nuclear staining problem.
> Your retrieval process isn't overly aggressive. I've used Vectors secondary
> and ABC reagents forever and have never had the issue you are describing.
> Human tissue?
>
> Jim Burchette, HT(ASCP) QIHC
> Histologist and Fly Fishing Bum
> Orlando, Florida
>
> 
> From: histonet-boun...@lists.utsouthwestern.edu [
> histonet-boun...@lists.utsouthwestern.edu] on behalf of Eva Permaul [
> e...@georgetown.edu]
> Sent: Tuesday, July 24, 2012 8:41 AM
> To: histonet@lists.utsouthwestern.edu
> Subject: Re: [Histonet] Secondary antibody causing nuclear staining?
>
> We standard use a Citrate pH6. We do 20min at 98C followed by cooling in
> the citrate for 20min.
> Eva
>
> On Tue, Jul 24, 2012 at 8:29 AM, James Burchette Jr. <
> james.burche...@duke.edu> wrote:
>
> > What is your heat retrieval process?
> >
> > Jim Burchette, HT(ASCP) QIHC
> > Histologist and Fly Fishing Bum
> > Orlando, Florida
> >
> > ____________
> > From: histonet-boun...@lists.utsouthwestern.edu [
> > histonet-boun...@lists.utsouthwestern.edu] on behalf of Eva Permaul [
> > e...@georgetown.edu]
> > Sent: Tuesday, July 24, 2012 8:13 AM
> > To: histonet@lists.utsouthwestern.edu
> > Subject: Re: [Histonet] Secondary antibody causing nuclear staining?
> >
> > I understand the point about the biotin and I should have said that when
> > using the ABC method we have taken to always using an avidin/biotin
> > blocking kit. We are using biotinylated secondary antibodies from
> Vector. I
> > have seen the same problem occur in our anti-mouse, anti-rabbit and
> > anti-goat. In my last run I had stomach fundus as well as skin melanoma,
> > both had pos.nuclei in the negative (no primary). In another run I had
> > colon ca and breast ca, the breast ca had fewer pos. nuclei than the
> colon
> > ca but they were still there. Some days the positive nuclei are stronger
> in
> > a sample that was just weakly positive before. Just want to understand
> what
> > it is and what effects it.
> > Thank you all for your ideas.
> > Eva Permaul
> > Georgetown University
> >
> > On Mon, Jul 23, 2012 at 7:16 PM, Tony Henwood (SCHN) <
> > tony.henw...@health.nsw.gov.au> wrote:
> >
> > > I should have added that this was from the workshop notes on a
> > > Hypotheticals Workshop I ran last year at our Australian National
> > Meeting.
> > >
> > > 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 Tony Henwood
> > > (SCHN)
> > > Sent: Tuesday, 24 July 2012 9:00 AM
> > > To: 'Eva Permaul'; histonet@lists.utsouthwestern.edu
> > > Subject: RE: [Histonet] Secondary antibody causing nuclear staining?
> > >
> > > It is possible that this is due to "Biotin nuclei" where excess biotin
> is
> > > found in the nuclei of some cells, see below:
> > >
> > > Optically clear nuclei have been reported in endometrial epithelium
> > > associated with first and second trimester abortions (Sickel & di
> > > Sant'Agnese 1994). Optically clear nuclei have also been found in
> > different
> > > types of tissues of diverse organs such as ovary, thyroid and lung
> > > (Nakatani et al 1994, Mount & Cooper 2001). The optically clear nuclei
> > > contain excess biotin.
> > >
> > > Endogenous biotin immunoreactivity is generally not visualized in
> > formalin
> > > fixed, paraffin-embedded tissues unless a heat-induced antigen
> retrieval
> > > step has been introduced (Mount & Cooper 2001).
> > >
> > > In this placental section, optically clear nuclei (containing biotin)
> > bind
> > > to the streptavidin of the ABC technique giv

Re: [Histonet] Secondary antibody causing nuclear staining?

[Eva Permaul]

We standard use a Citrate pH6. We do 20min at 98C followed by cooling in
the citrate for 20min.
Eva

On Tue, Jul 24, 2012 at 8:29 AM, James Burchette Jr. <
james.burche...@duke.edu> wrote:

> What is your heat retrieval process?
>
> Jim Burchette, HT(ASCP) QIHC
> Histologist and Fly Fishing Bum
> Orlando, Florida
>
> 
> From: histonet-boun...@lists.utsouthwestern.edu [
> histonet-boun...@lists.utsouthwestern.edu] on behalf of Eva Permaul [
> e...@georgetown.edu]
> Sent: Tuesday, July 24, 2012 8:13 AM
> To: histonet@lists.utsouthwestern.edu
> Subject: Re: [Histonet] Secondary antibody causing nuclear staining?
>
> I understand the point about the biotin and I should have said that when
> using the ABC method we have taken to always using an avidin/biotin
> blocking kit. We are using biotinylated secondary antibodies from Vector. I
> have seen the same problem occur in our anti-mouse, anti-rabbit and
> anti-goat. In my last run I had stomach fundus as well as skin melanoma,
> both had pos.nuclei in the negative (no primary). In another run I had
> colon ca and breast ca, the breast ca had fewer pos. nuclei than the colon
> ca but they were still there. Some days the positive nuclei are stronger in
> a sample that was just weakly positive before. Just want to understand what
> it is and what effects it.
> Thank you all for your ideas.
> Eva Permaul
> Georgetown University
>
> On Mon, Jul 23, 2012 at 7:16 PM, Tony Henwood (SCHN) <
> tony.henw...@health.nsw.gov.au> wrote:
>
> > I should have added that this was from the workshop notes on a
> > Hypotheticals Workshop I ran last year at our Australian National
> Meeting.
> >
> > 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 Tony Henwood
> > (SCHN)
> > Sent: Tuesday, 24 July 2012 9:00 AM
> > To: 'Eva Permaul'; histonet@lists.utsouthwestern.edu
> > Subject: RE: [Histonet] Secondary antibody causing nuclear staining?
> >
> > It is possible that this is due to "Biotin nuclei" where excess biotin is
> > found in the nuclei of some cells, see below:
> >
> > Optically clear nuclei have been reported in endometrial epithelium
> > associated with first and second trimester abortions (Sickel & di
> > Sant'Agnese 1994). Optically clear nuclei have also been found in
> different
> > types of tissues of diverse organs such as ovary, thyroid and lung
> > (Nakatani et al 1994, Mount & Cooper 2001). The optically clear nuclei
> > contain excess biotin.
> >
> > Endogenous biotin immunoreactivity is generally not visualized in
> formalin
> > fixed, paraffin-embedded tissues unless a heat-induced antigen retrieval
> > step has been introduced (Mount & Cooper 2001).
> >
> > In this placental section, optically clear nuclei (containing biotin)
> bind
> > to the streptavidin of the ABC technique giving a reaction similar to
> that
> > seen with CMV containing cells. If a polymer method (or even the original
> > Sternberger's PAP method) is used then this anomalous staining will
> > disappear, thus allowing confident demonstration of CMV infected nuclei.
> >
> > The false-positive staining pattern caused by endogenous biotin can be
> > cytoplasmic or nuclear. A report of positive immunoreactivity of
> > hepatocellular carcinomas for inhibin was later determined to be a
> > false-positive finding due to cytoplasmic endogenous biotin. Steroid cell
> > tumours of the ovary were found to demonstrate endogenous biotin
> > cytoplasmic staining in 36% of cases. Immunoreactivity for anti-Herpes
> > virus immunohistochemical staining in a series of endometria was also
> later
> > determined to be a false-positive result due to biotin. The prominent
> > intranuclear inclusions, resembling herpes virus cytopathic effect, were
> > caused by intranuclear biotin and not viral particles. Similar false
> > positive staining for CMV in products of conception has also been
> reported
> > (Mount & Cooper 2001).
> >
> > False-positive staining can be cytoplasmic or nuclear. When cytoplasmic,
> > the appearance of the false signal is that 

Re: [Histonet] Secondary antibody causing nuclear staining?

[Eva Permaul]

I understand the point about the biotin and I should have said that when
using the ABC method we have taken to always using an avidin/biotin
blocking kit. We are using biotinylated secondary antibodies from Vector. I
have seen the same problem occur in our anti-mouse, anti-rabbit and
anti-goat. In my last run I had stomach fundus as well as skin melanoma,
both had pos.nuclei in the negative (no primary). In another run I had
colon ca and breast ca, the breast ca had fewer pos. nuclei than the colon
ca but they were still there. Some days the positive nuclei are stronger in
a sample that was just weakly positive before. Just want to understand what
it is and what effects it.
Thank you all for your ideas.
Eva Permaul
Georgetown University

On Mon, Jul 23, 2012 at 7:16 PM, Tony Henwood (SCHN) <
tony.henw...@health.nsw.gov.au> wrote:

> I should have added that this was from the workshop notes on a
> Hypotheticals Workshop I ran last year at our Australian National Meeting.
>
> 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 Tony Henwood
> (SCHN)
> Sent: Tuesday, 24 July 2012 9:00 AM
> To: 'Eva Permaul'; histonet@lists.utsouthwestern.edu
> Subject: RE: [Histonet] Secondary antibody causing nuclear staining?
>
> It is possible that this is due to "Biotin nuclei" where excess biotin is
> found in the nuclei of some cells, see below:
>
> Optically clear nuclei have been reported in endometrial epithelium
> associated with first and second trimester abortions (Sickel & di
> Sant'Agnese 1994). Optically clear nuclei have also been found in different
> types of tissues of diverse organs such as ovary, thyroid and lung
> (Nakatani et al 1994, Mount & Cooper 2001). The optically clear nuclei
> contain excess biotin.
>
> Endogenous biotin immunoreactivity is generally not visualized in formalin
> fixed, paraffin-embedded tissues unless a heat-induced antigen retrieval
> step has been introduced (Mount & Cooper 2001).
>
> In this placental section, optically clear nuclei (containing biotin) bind
> to the streptavidin of the ABC technique giving a reaction similar to that
> seen with CMV containing cells. If a polymer method (or even the original
> Sternberger's PAP method) is used then this anomalous staining will
> disappear, thus allowing confident demonstration of CMV infected nuclei.
>
> The false-positive staining pattern caused by endogenous biotin can be
> cytoplasmic or nuclear. A report of positive immunoreactivity of
> hepatocellular carcinomas for inhibin was later determined to be a
> false-positive finding due to cytoplasmic endogenous biotin. Steroid cell
> tumours of the ovary were found to demonstrate endogenous biotin
> cytoplasmic staining in 36% of cases. Immunoreactivity for anti-Herpes
> virus immunohistochemical staining in a series of endometria was also later
> determined to be a false-positive result due to biotin. The prominent
> intranuclear inclusions, resembling herpes virus cytopathic effect, were
> caused by intranuclear biotin and not viral particles. Similar false
> positive staining for CMV in products of conception has also been reported
> (Mount & Cooper 2001).
>
> False-positive staining can be cytoplasmic or nuclear. When cytoplasmic,
> the appearance of the false signal is that of a dull brown granular or
> fluffy staining pattern. If this quality of staining is observed with
> several different antibodies, endogenous staining by biotin should be
> considered. When nuclear, a false-positive reaction may be associated with
> optically clear nuclei identified on H&E stained sections. False-positive
> staining due to endogenous biotin, however, does not occur in a cell
> membrane pattern (Mount & Cooper 2001).
>
> Mount SL & Cooper K (2001) "Beware of biotin: a source of false-positive
> immunohistochemistry" Current Diagnostic Pathology  7:161-167.
> Nakatani et al (1994) Am J Surg Pathol 18(6):637-642.
> Sickel & di Sant'Agnese (1994) Arch Pathol Lab Med 118:831-833
>
>
> 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, AUST

Re: [Histonet] Secondary antibody causing nuclear staining?

[Eva Permaul]

We believe it has to be the secondary as we have nuclear staining in the
absence of primary antibody.
Eva

On Mon, Jul 23, 2012 at 12:58 PM, ihcs-wojcieszyn  wrote:

> Hello,
> Please explain how you know that it is the secondary and not the
> primary? We Have confirmed that in canine lymphomas, nuclear
> CD79a is a real and frequent observation.
>
> john
> IHC Services
>
> On Monday, July 23, 2012, at 08:39 AM, Eva Permaul wrote:
>
>  Hello,
>>
>> I have noticed that our biotinylated secondary antibodies on occasion
>> cause
>> nuclear staining in some samples. Why is this? It is not every time so I
>> find it rather stange. Anyone know why this is happening and what I can do
>> to avoid it?
>>
>> Thank you for any suggestion,
>> Eva Permaul
>> Georgetown University
>> __**_
>> Histonet mailing list
>> Histonet@lists.utsouthwestern.**edu 
>> http://lists.utsouthwestern.**edu/mailman/listinfo/histonet<http://lists.utsouthwestern.edu/mailman/listinfo/histonet>
>>
>>
>
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

[Histonet] Secondary antibody causing nuclear staining?

[Eva Permaul]

Hello,

I have noticed that our biotinylated secondary antibodies on occasion cause
nuclear staining in some samples. Why is this? It is not every time so I
find it rather stange. Anyone know why this is happening and what I can do
to avoid it?

Thank you for any suggestion,
Eva Permaul
Georgetown University
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

[Histonet] Looking for a published PLAP antibody that is not made in mouse

[Eva Permaul]

Good afternoon,
I have now spent way too much time trying to find a placental-like 
alkaline phosphatase antibody that is not made in mouse and has been 
published with pretty IHC-P pictures. All I keep finding are made in 
mouse. Could someone please direct me to a good one? I have found lots 
of rabbit made PLAP antibodies from companies but the investigator wants 
me to find one that has also been published.

Thank you all for your help,
Eva Permaul
Georgetown University

___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

[Histonet] Counterstain for dual chromogenic

[Eva Permaul]

Good morning,
I have been attempting to do some dual chromogenic staining. I am using 
a mouse and a rabbit antibody. For the mouse antibody I am using an 
HRP-mouse secondary followed by AEC for visualization. For the rabbit 
antibody I am using a biotinylated rabbit secondary, followed by ABC-AP 
and vector blue. My problem is what to use as a counterstain. From 
everything I have read so far there isn't one that would work without 
having some problems. I was thinking of trying a very light Hematoxylin 
and see if it doesn't disrupt the vector blue visualization too much. 
Does anyone else have any suggestions?

Thanks,
Eva

___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

[Histonet] dehydration of hydrated slide....

[Eva Permaul]

Good morning,
I accidentally hydrated a FFPE slide that I can not stain today. It is 
in water. I did not do antigen retrieval. What do I do? Can I dehydrate 
the slide again? Will I be able to stain it later if I do?

Thanks,
Eva Permaul
Georgetown University


___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

[Histonet] EGFR antibody for staining mouse tissue?

[Eva Permaul]

Hello,
Does anybody use an EGFR antibody for staining on mouse tissue? Is it a 
published antibody? Would you be willing to share your protocol?

Thanks,
Eva
Georgetown University

___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

[Histonet] silly question about goat serum

[Eva Permaul]

Good morning,
I have a question that is probably very silly but I just don't want to 
make any mistakes with the experiment I am suppose to do. I am used to 
the protein blocking being called normal goat serum (10%). The kit I am 
about to use says their's is 10% goat non-immune serum. Is this the same 
thing?

Thanks for your help of a confused tech,
Eva Permaul
Georgetown University

___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

[Histonet] avidin/biotin blocking???

[Eva Permaul]

Good afternoon out there in histoland,
I am trying to find a cheaper alternative to the avidin/biotin blocking 
system we are using now so that we can use it for larger bulk staining 
cases. Right now we only use it for small hand staining cases.
I read somewhere that there are those who make these reagents 
themselves. Would anyone be willing to share their recipes and how do 
they compare to company bought ones?

Thanks for your help,
Eva Permaul
Georgetown University

___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

[Histonet] staining rat tissue with monoclonal antibody?

[Eva Permaul]

Good afternoon.
I just had someone ask me about staining rat tissue using an monoclonal 
(mouse) antibody. I have never done this before and so was wondering if 
there is anything in particular I need to keep in mind. Does mouse 
antibodies cause a problem when staining rat tissues? Do I have to use a 
specific kit or specific secondary antibody? Can I use a biotinylated 
goat-anti-mouse secondary followed by ABC?

Thanks for your help,
Eva

___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

[Histonet] bluing in tap water?

[Eva Permaul]

Good morning,
I was wondering if someone uses tap water to blue their slides after 
Hematoxyline. If yes, do you use warm or cold water and for how long?

Thanks,
Eva
Georgetown University

___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

[Histonet] bluing hematoxylin and alkaline water????

[Eva Permaul]

Good morning out in histoland,
Thank god it is Friday. Have just a small question that I am sure you 
all have an answer for.
It is about bluing slides after hematoxylin. I read somewhere that 
alkaline water can blue hematoxylin and I am wondering if this is part 
of our problem. We use an automated stainer that we run both during the 
day and during the night. Normally we blue the slides in 1% ammonium 
after the runs for 1min. The run that was done during the day ended up 
less blue (more purple). The ones that run during the night continue 
being washed in water until we come in in the morning. They ended up 
more blue. I tested the pH of the water we are using. It is 7.96. Would 
this be enough to blue the slides if they are washed in it every hour 
from 9pm to 8am? Any other reason this might be happening? Should we not 
blue the slides that are run during the night? Why do we blue 
hematoxylin anyways?

Thank you for all your answers
Eva Permaul
(still learning)

___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

[Histonet] gastrin antibodies.....

[Eva Permaul]

Good morning out there in histoland,
I am looking for some help in finding two antibodies:
- sulfated gastrin-17 (G-7)
- sulfated cholecystokinin-8 (CCK-8)
All the antibodies that I are finding are non-sulfated.
Thank you all for your help,
Eva Permaul
Georgetown University

___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet