[Histonet] Penetration problem with floating brain sections

[Michelle Chang]

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-request at 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)
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> ----------------------------------------------------------------------
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> Message: 1
> Date: Thu, 16 Feb 2017 20:13:33 +0000
> From: "Johnson, Carole" <cjohnson at nmda.nmsu.edu>
> To: "histonet at lists.utsouthwestern.edu"
>    <histonet at lists.utsouthwestern.edu>
> Subject: [Histonet] Opening in great veterinary diagnostic lab
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> 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
> 
> 
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> ------------------------------
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> Message: 2
> Date: Fri, 17 Feb 2017 06:56:25 +0000
> From: "Bass, Caroline" <cebass at buffalo.edu>
> To: "histonet at lists.utsouthwestern.edu"
>    <histonet at lists.utsouthwestern.edu>
> Subject: [Histonet] penetration problem with floating brain sections
> Message-ID: <DC7C5694-2B50-472B-B4DB-FE69E89E0481 at buffalo.edu>
> 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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