[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:
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> Today's Topics:
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>   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 
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> 
> --
> 
> 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   
> 
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> End of Histonet Digest, Vol 159, Issue 15
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[Histonet] MOHS embedding

[Victoria Kline via Histonet]

Hello! I've been a histotech for several years and have recently joined a 
dermatology practice that has started doing MOHS. I was curious which embedding 
technique most MOHS techs prefer to use and why? Currently I've been using a 
heat sink (not attached) to do my embedding. We only have one heat sink, so 
I'll do my embedding on the free heat sink and then flip that "button" over 
onto a chuck with semi solid embedding medium. I don't let anything set on top 
(since we don't have another heat sink). I've done a lot of research on the 
cryoembedder, but its very expensive and my practice won't go for the cost. I'm 
interested in trying the slide technique, but wanted to hear from other techs 
about it. Do you put any embedding medium on the slide first or not? I've seen 
it described both ways. Any feedback is appreciated, especially on which 
technique has given the best results for speed and accuracy on full epidermis. 

Victoria Kline HT (ASCP)
Lancaster, PA
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[Histonet] ultraclone RA95101 PGP 9.5 aby source or replacement

[Travers, Susan via Histonet]

Hello,
Does anyone know a good replacement for the anti PGP 9.5 aby offered by 
Ultraclone. This seems to have been the most common source for this aby for 
many years but I cannot find a website in order to buy it so assume it's no 
longer available.  Any advice would be greatly appreciated.

We would like to stain nerve fibers in mouse and human tongue.

Thanks!

st
Susan Travers
Professor
Division of Biosciences
College of Dentistry
The Ohio State University

305 West 12th Avenue
Columbus, Ohio  43210

614-292-6366 (office)
614-457-6945 (fax)
614-361-0800 (cell)

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