Re: Help!!!!!!!!! Embedding cells....urgent!!!

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From:Paul Klosen <>
To:Emma Carter <> (by way of Histonet),
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At 08:39 17/05/00 -0500, Emma Carter a écrit:

>Sometimes, it would be really nice to have some notice of these things!!!!
>Afetr i have severely castigated the relevant parties, please can someone
>I wrote a while ago about embedding cells in paraffin.....Problem is,
>collegue has started them growing today and is going to harvest on friday. I
>am on holiday on friday afternoon, which means i have zero time on friday
>morning to do anything with these cells.
>So, i have loads of panic driven questions!!!
>1) If i pellet them and put them into a gelatine capsule, will they be okay
>in the fridge freezer over the weekend?

I'd pellet them in an eppendorf tube to leave over the weekend.

>2) I cant get any histogel in time, so can someone let me have a suitable
>recipe for gelatine???

Try using agarose. Gelatin tends to become pretty hard upon dehydration and=20
embedding. With one lot of gelatin that I used, I ended up with blocks 
about the hardness of bone !!
For agarose, make up a 1% solution in buffer (2% if you are using 
agar-agar). Heat until complete dissolution of the agarose (usually needs 
boiling), then cool down in waterbath to about 55-60°C.
Preheat your FIXED cells to the same temperature, then transfer into the 
warm agarose for 30 min in an eppendorf tube. Spin the cells down for a few=20
minutes and let the agarose harden in the eppendorf tube.
To take your pellet out, cut of the tip of the eppendorf tube with a 
scalpel blade. You need only cut a small hole into the tip in order to let=20
the air gel below the agarose. Recover the cone of agarose with your cells=20
and block to the appropriate size. Dehydrate and embedd.

>3)Should i fix them in NBF or 4% Para?

Depends on what you want to do with your cells.

>4)Should i fix them before embedding them in gelatine?

Definitely, whether you use gelatine, agarose or histogel, fix the cells 

>5) Can i leave them in fixative over the weekend?

Depends on what you want to do with your cells. For ICC, I don't think it 
would be a good idea. What you can do is simply fix the cells on friday (or=20
tell your colleague how to do it), rinse in buffer and leave the cells in 
buffer over the weekend. Process on monday.

>6) can i go home and cry!?

If you want to ... ;o)

                                                                        (o -) O
Paul Klosen, PhD
CNRS UMR 7518 Neurobiologie des Fonctions Rythmiques et Saisonnieres
Universite Louis Pasteur  12, rue de l'Universite
F-67000 Strasbourg, FRANCE
Tel.  Fax.

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