[3view] 3view Digest, Vol 26, Issue 1

Jokitalo, Eija eija.jokitalo at helsinki.fi
Wed Dec 7 12:05:53 CET 2016


Hi,
We have been testing pre-embedding immunolabelling of cultured cells with some success. The protocol for embedding is the same we routinely use except we omit uranyl acetate staining. As you can see, images are noisy, but otherwise pretty nice. We have used PLP-fixation, saponin permeabilization, secondary antibodies conjugated with 1.4 nm nanogold particles and silver enhancement (Nanoprobes kit) followed by gold toning with self-made solutions.
I hope the attached video works. 

Best Wishes, Eija



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Today's Topics:

   1. immunolabelling (Kathryn White)
   2. Re: immunolabelling (Minh Huynh)
   3. Re: immunolabelling (Louise Hughes)


----------------------------------------------------------------------

Message: 1
Date: Mon, 5 Dec 2016 13:13:11 +0000
From: Kathryn White <kathryn.white at newcastle.ac.uk>
To: "3view at open-em.org" <3view at open-em.org>
Subject: [3view] immunolabelling
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	<VI1PR0701MB28006B89E919170233114D3FAC830 at VI1PR0701MB2800.eurprd07.prod.outlook.com>
	
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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






------------------------------

Message: 2
Date: Tue, 6 Dec 2016 00:39:55 +0000
From: Minh Huynh <minh.huynh at sydney.edu.au>
To: Mailing list for Gatan 3View Serial Block-Facing
	<3view at open-em.org>
Subject: Re: [3view] immunolabelling
Message-ID: <D46C5213.114B4%minh.huynh at sydney.edu.au>
Content-Type: text/plain; charset=WINDOWS-1252

Hi Kath,

A couple of my users have tried to image immuno-gold labelled cells on the 3View. We?ve 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 don?t 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.

I?d be very interested to hear about other people?s experience and success or otherwise.

Minh


DR MINH HUYNH | General Microscopist
Australian Centre for Microscopy & Microanalysis

THE UNIVERSITY OF SYDNEY
Rm 124, Madsen Building F09 | The University of Sydney | NSW | 2006 | Australia T +61 2 9351 7525 | F +61 2 9351 7682 E minh.huynh at sydney.edu.au | W http://sydney.edu.au/acmm

My working days are Monday, Tuesday, Wednesday & Friday

Incorporating:
Australian Microscopy & Microanalysis Research Facility (AMMRF) | W http://www.ammrf.org.au <http://www.ammrf.org.au/>

CRICOS 00026A
This email plus any attachments to it are confidential. Any unauthorised use is strictly prohibited. If you receive this email in error, please delete it and any attachments.





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
>Framlington Place
>Newcastle University
>Newcastle upon Tyne
>NE2 4HH
>
>0191 2086966
>
>http://www.ncl.ac.uk/emrs
>
>
>
>
>_______________________________________________
>3view mailing list
>3view at open-em.org
>http://open-em.org/mailman/listinfo/3view
>



------------------------------

Message: 3
Date: Tue, 6 Dec 2016 08:57:33 +0000
From: Louise Hughes <lhughes at brookes.ac.uk>
To: Mailing list for Gatan 3View Serial Block-Facing
	<3view at open-em.org>
Subject: Re: [3view] immunolabelling
Message-ID:
	<CAP_aGBRh6++AxJk9i8vsFg+jU9oSXCrbVBVOy3oPH8b5Moqk7g at mail.gmail.com>
Content-Type: text/plain; charset="utf-8"

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. We?ve 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 don?t 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.
>
> I?d be very interested to hear about other people?s experience and 
> success or otherwise.
>
> Minh
>
>
> DR MINH HUYNH | General Microscopist
> Australian Centre for Microscopy & Microanalysis
>
> THE UNIVERSITY OF SYDNEY
> Rm 124, Madsen Building F09 | The University of Sydney | NSW | 2006 | 
> Australia T +61 2 9351 7525 | F +61 2 9351 7682 E 
> minh.huynh at sydney.edu.au | W http://sydney.edu.au/acmm
>
> My working days are Monday, Tuesday, Wednesday & Friday
>
> Incorporating:
> Australian Microscopy & Microanalysis Research Facility (AMMRF) | W 
> http://www.ammrf.org.au <http://www.ammrf.org.au/>
>
> CRICOS 00026A
> This email plus any attachments to it are confidential. Any 
> unauthorised use is strictly prohibited. If you receive this email in 
> error, please delete it and any attachments.
>
>
>
>
>
> 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
> >Framlington Place
> >Newcastle University
> >Newcastle upon Tyne
> >NE2 4HH
> >
> >0191 2086966
> >
> >http://www.ncl.ac.uk/emrs
> >
> >
> >
> >
> >_______________________________________________
> >3view mailing list
> >3view at open-em.org
> >http://open-em.org/mailman/listinfo/3view
> >
>
> _______________________________________________
> 3view mailing list
> 3view at open-em.org
> http://open-em.org/mailman/listinfo/3view
>



--
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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