[3view] immunolabelling

Louise Hughes lhughes at brookes.ac.uk
Tue Dec 6 09:57:33 CET 2016


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> 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
>
>
> DR MINH HUYNH | General Microscopist
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> On 6/12/2016 12:13 am, "Kathryn White" <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
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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
Personal website: http://www.miniaturehorizons.com
Twitter: https://twitter.com/DrLouiseCHughes
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