Emma,
	             I did something similar with CHO cells for paraffin. We took the Pelleted cells in a 15 ml tube (I tried 0.5,1and 10 million cells/ml) we fixed in 10%NBF (you could fix then spin down we didn't ) for 24hr or 3 days. After fixation the fixative was aspirated off and replaced with PBS and washed 3 times.  The pellet was transferred to a 1.5-ml microcentrifuge (tube,soft easy to cut) by freezing the pellet that is in the 15 ml hard tube with liquid nitrogen (place 15 ml tube in liquid nitrogen about 20 seconds is enough) and detaching the pellet from the bottom (by hitting it on the bench )of the conical tube. The cells (frozen) are quickly placed in the 1.5 ml tube and surrounded by gelatin.  Cells were infiltrated w/ gelatin at this point the pellet starts to thaw, so work quickly and that's why in my hands it was better to have more cells 1- 10 million. The gelatin hardened at -20 degrees C in about 15-20 minutes. The gelatin capsule could be cut out of the centrifuge tube, wrapped in lens paper, cassetted and run in the VIP.  The bigger the pellet the easier it was to see the pellet after processing. Most of the gelatin was gone after processing but you can still see the bigger pellets to embed them. You could pellet the cells in the 1.5 tube but I got the pellet in the 15ml. The only thing is that after detaching the pellet I could suspend the pellet in the gelatin in the 1.5 tube so there was no air pockets and once hardened I could cut right through the tube on either side of the pellet an push the gelatin/pellet  out onto the lens paper. The cells looked good and I did IHC on them (it worked).......Good luck.





Jamie Erickson
Associate Scientist
Genetics Institute
1 Burtt Rd.
Andover, MA   01810
work : (978) 247-1348
FAX  : (978) 247-1333

>>> Emma Carter <E.Carter@OxfordBioMedica.co.uk> 04/26 11:40 AM >>>

I am sure i read a message about this recently, but i cant recall, and i
dont have anything in my mail folder...

someone has asked me about paraffin embedding some cells. I came up with a
protocol off the top of my head, roughly:

Put cells in eppendorf tube, spin down and remove culture buffer.
Resuspend with formalin and leave for 10 mins. Spin down and remove
formalin.
Resuspend/spin down through serial alcohols, then xylene.
after final resuspension with xylene, remove and cover with warm wax.
Allow wax pellet to set. Pop out of tube and embed in cassette.

How does this appear to other people? Should i go through the dehydration
steps?

Any help would be very appreciated...... (oh yeah, this is all to be done by
my own fair hand.......automation!? Pah!)

Thanks in advance,

emma carter