[Histonet] Penetration problem with floating brain sections

[Michelle Chang via Histonet]

Hi Caroline,

For floating sections of 50um, my primary antibody incubation has always been 
overnight at room temp -- much better staining.  You may also want to add 
Triton-X or Tween 20 to your wash buffer (in case you have not done so).

In regards to your slide cracking, where do you observe the crack?  If it is 
peripheral and localized on one side, you may want to check the angle of the 
blade.  If it is throughout, then you may want to check if block has totally 
sink in sucrose prior to sectioning.  

Let me know if you have any additional question.

Michelle

> On Feb 17, 2017, at 10:00 AM, histonet-requ...@lists.utsouthwestern.edu wrote:
> 
> Send Histonet mailing list submissions to
>histonet@lists.utsouthwestern.edu
> 
> To subscribe or unsubscribe via the World Wide Web, visit
>http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> or, via email, send a message with subject or body 'help' to
>histonet-requ...@lists.utsouthwestern.edu
> 
> You can reach the person managing the list at
>histonet-ow...@lists.utsouthwestern.edu
> 
> When replying, please edit your Subject line so it is more specific
> than "Re: Contents of Histonet digest..."
> 
> 
> Today's Topics:
> 
>   1. Opening in great veterinary diagnostic lab (Johnson, Carole)
>   2. penetration problem with floating brain sections (Bass, Caroline)
> 
> 
> --
> 
> Message: 1
> Date: Thu, 16 Feb 2017 20:13:33 +
> From: "Johnson, Carole" 
> To: "histonet@lists.utsouthwestern.edu"
>
> Subject: [Histonet] Opening in great veterinary diagnostic lab
> Message-ID:
>
> Content-Type: text/plain; charset="us-ascii"
> 
> We have a day shift opening in our lab. Beautiful lab, great equipment, 
> awesome pathologists.  If you are interested, please follow this link for 
> more information.
> 
> http://www.nmda.nmsu.edu/humanresources/
> 
> 
> 
> Carole L. Johnson, HT(ASCP)cm, QIHC
> 
> New Mexico Department of Agriculture
> Veterinary Diagnostic Services
> 1101 Camino de Salud, NE
> Albuquerque, NM 87101
> 505.383.9299
> 
> 
> 
> 
> Confidentiality Notice: New Mexico has a very broad public records law. Most 
> written communications to or from state employees are public records. Your 
> e-mail communications may therefore be subject to public disclosure. This 
> e-mail, including all attachments is for the sole use of the intended 
> recipients. Any unauthorized review, use, disclosure or distribution is 
> prohibited unless specifically provided under the New Mexico Inspection of 
> Public Records Act.
> 
> 
> --
> 
> Message: 2
> Date: Fri, 17 Feb 2017 06:56:25 +
> From: "Bass, Caroline" 
> To: "histonet@lists.utsouthwestern.edu"
>
> Subject: [Histonet] penetration problem with floating brain sections
> Message-ID: 
> Content-Type: text/plain; charset="utf-8"
> 
> Hello All, 
> 
> I?m doing some IHC with DAB on formaldehyde fixed rat brain floating 
> sections, either 35 of 50 um thick. Usually I get fine staining of TH for 
> example, but now I?m going after a neuron that's a bit sparse, and I noticed 
> I have a couple of darkly stained cells. But I can see dozens of ?ghost 
> cells?, these appear to be very lightly stained in the right place. My guess 
> is that the antibody isn?t penetrating fully, and so cells on the face of the 
> sections are staining darkly, and some on the interior are just barely been 
> stained. Any suggestions on improving penetrance? I usually have primary on 
> overnight, shaking in the cold room, and secondary at RT for 2 hours. I was 
> thinking of extending the primary to room temperature and maybe upping the 
> triton-X concentration. Any other suggestions?
> 
> Secondly, I noticed were getting some cracks in the tissue. We usually mount 
> the DAB stained sections on some sort of tissue bond slide, and then let it 
> dry overnight before dilipidating and coverslipping with permeant. My best 
> guess is that sometime during the drying or coverslipping the tissue pulls 
> away slight, resulting in these smallish cracks.
> 
> Anyway, any advice on handling these two issues would be greatly appreciated.
> 
> Thanks,
> 
> Caroline Bass
> University at Buffalo   
> 
> --
> 
> Subject: Digest Footer
> 
> ___
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> 
> --
> 
> End of Histonet Digest, Vol 159, Issue 15
> *

___
Histonet mailing list
Histonet@lists.utsouthwestern.edu

[Histonet] Chicken samples

[Michelle Chang via Histonet]

Hi,

The reactivity of the antibodies depend on the epitope/immunogen of the
antibodies. I would advice to check % homology of the eptiope/immunogen to
that of chicken to know if the antibodies work for on chicken.  High
homology means it is very much likely that it will work.

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

[Histonet] PFA Fixed tissue not sticking to slides?

[Michelle Chang via Histonet]

Good charged slides or subbed slides will work.

In regards to the fixation, I personally find the morphology with 4% PFA O/N 
followed by 30% sucrose better than doing them simultaneously.

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

[Histonet] ARC immunohistochemistry- "sticky sections" in DAB

[Michelle Chang via Histonet]

Hi Gabrielle,

Are you doing floating sections?  Did you cut the tissue recently?  Do you
notice your tissue is also "sticky"? If you do, there is a possibility that
the tissue may be infected by bacteria. Try washing the tissue with PBS or
TBS with 0.1% sodium azide a couple time. Sometimes it help.

Good Luck

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