[3view] immunolabelling

Miller, Marcia MMiller at coh.org
Tue Dec 6 20:39:28 CET 2016


Hi Everyone,

It is nice to have this connection to other 3View labs.

I have another question looking for experience with using the 3View to section cells on different kinds of supports like nylon filters and different scaffolds used in bioengineering.  We will soon begin projects in which we will need to image cells grown on supports.  Not sure if this will be easier than making sections, but it might be.

Any comments?

Marcia Miller

Director, EM/AFM Core Facility
Beckman Research Institute, City of Hope
1500 E Duarte Road | Duarte, CA 91010-3000
t 626.301.8264 | mamiller at coh.org<mailto:mamiller at coh.org>



From: 3view-bounces at open-em.org [mailto:3view-bounces at open-em.org] On Behalf Of Louise Hughes
Sent: Tuesday, December 06, 2016 12:58 AM
To: Mailing list for Gatan 3View Serial Block-Facing
Subject: Re: [3view] immunolabelling

Dear all,

This is something we will be attempting soon, but have not yet done so. I have done SEM immunolabelling in the past (not with a serial sectioning technique) and we used pre-embedding immunolabelling with 5nm gold followed by gold enhancement of the labels. We were interested in the cytoskeleton and the cells were permeabilised prior to labelling. From memory, the gold enhancement was tricky to optimise but worked very well once that had been achieved. It may be a technique that could be used with 3View where resolution is limited compared with TEM. Our protocol is outlined in this paper http://www.ecmjournal.org/journal/papers/vol004/pdf/v004a01.pdf and the work done to optimise the enhancement is referenced.

Regards,
Louise

On 6 December 2016 at 00:39, Minh Huynh <minh.huynh at sydney.edu.au<mailto:minh.huynh at sydney.edu.au>> wrote:
Hi Kath,

A couple of my users have tried to image immuno-gold labelled cells on the
3View. We¹ve not had much success but this is mainly due to sample prep
rather than the 3View it self. We did pre-embedding labelling and the cell
ultrastructure looked awful! We still need to work on this as I don¹t have
that much experience with immuno-gold, however, my gut feeling tells me
that serial section SEM or TEM would be better for this. At best, we are
getting pixel sizes of between 3 - 5 nm on the 3View which I think is a
bit of a stretch to see 10 nm gold.

I hope that helps.

I¹d be very interested to hear about other people¹s experience and success
or otherwise.

Minh


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On 6/12/2016 12:13 am, "Kathryn White" <kathryn.white at newcastle.ac.uk<mailto:kathryn.white at newcastle.ac.uk>>
wrote:

>Dear All
>Has anyone tried any immunolabelling - either DAB or gold - in
>combination with the 3view? I'd be interested to hear about unsuccessful
>as well as successful attempts!
>Thanks
>Kath
>
>Dr Kath White
>EM Research Services
>Faculty of Medical Sciences
>Framlington Place
>Newcastle University
>Newcastle upon Tyne
>NE2 4HH
>
>0191 2086966
>
>http://www.ncl.ac.uk/emrs
>
>
>
>
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--
Dr. Louise Hughes
Bio-Imaging Unit Researcher and  Microscopist
Department of Biological and Medical Sciences,
Oxford Brookes University,
Oxford, OX3 0BP, UK
01865 484351

Brookes e-mail: lhughes at brookes.ac.uk<mailto:lhughes at brookes.ac.uk>
Personal website: http://www.miniaturehorizons.com
Twitter: https://twitter.com/DrLouiseCHughes




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