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Sphere staining protocol (agarose)


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#1 Rute

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Posted 15 February 2012 - 10:16 AM

Hey,

I am growing putative stem cell spheres and I would like to use them for immunofluorescence.
I have seen many different ways of doing it. Some just put the sphere onto the slide, others include the spheres in agarose and them either freeze it and ultra cut or include them in parafin and then cut them.

Is anyone staining spheres that would be kind enough to share somne thoughts.
What technique should I use?

Thanks,
Rute

#2 Tabaluga

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Posted 15 February 2012 - 10:48 AM

The problem with a sphere on a slide is that since it is three-dimensional, you will almost for sure have several layers of cells. Are you planning to use a confocal microscope ? Then it might work.

Embedding the spheroids in agarose is the better option if you don't use confocal, but obviously it takes rather longer.

Edited by Tabaluga, 15 February 2012 - 10:49 AM.

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#3 Rute

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Posted 16 February 2012 - 02:39 AM

I was thinking of going for the agarose protocol.
Thanks for the help.
Do you have the protocol for that? I am starting this work int he lab so no one has an idea on how to do it.
Can I kindly ask you for your advice?

Thank you

#4 Tabaluga

Tabaluga

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Posted 16 February 2012 - 04:29 AM

Unfortunately, I don't have an agarose protocol... The only thing I have is a protocol for embedding spheroids in a collagen gel, but I haven't yet tried it out. The protocol I know can be found here:

Google Books => "Methods in endothelial cell biology" by Hellmut G. Augustin => starting from page 115

They do it with endothelial cell spheroids, but the basic protocol should be applicable to stem cell spheres too. It's with a collagen gel, though.

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 





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