Nick, I am assuming that your 3D cells only grow on plastic. They make plastic cover slips which, if your cells attach to them and grow, might make the embedding much easier. You would follow the same method as stated, but then you could invert the coverslips on a beem capsule and separate the coverslip from capsule with liquid nitrogen. However, I have never done it with plastic coverslips (only glass), so not sure if they would easily separate from capsule with liquid nitrogen. If anyone else has done so, please weigh in.
Peggy -----Original Message----- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Geoff McAuliffe Sent: Wednesday, January 27, 2010 3:20 PM To: Nicholas David Evans Cc: histonet@lists.utsouthwestern.edu Subject: Re: [HISTONET] embedding cell cultures Hi Nick: You can use the Epon substitutes such as EmBed 812. Fix, osmicate, dehydrate as usual, but omit the proplylene oxide as it will react with the plastic dish. Epon substitutes will mix with ethanol. I used 2:1 then 1:1 then 1:2 ratios of absolute ethanol to "Epon" with catalyst added with agitation. Then several changes of pure Epon and polymerize. Yes, you will have to saw away the plastic, if you try to section the Epon-plastic combo the two will separate. How much easier do you want it to be? Geoff Nicholas David Evans wrote: > Dear all, > > I was hoping someone might be able to offer me some advice on embedding and sectioning cell cultures. > > In short we are growing cells which form 3D dome-like structures on tissue culture plastic. Does anyone have any experience or advice to offer on embedding the cultures in situ before sectioning? I have seen various methods in the literature, which often use Epon to embed the material followed by sawing away the plastic, but if anyone can offer some tips on other possible (easier) ways of doing it, or can refer me to some useful literature, I'd be very grateful. > > I would like to have simple 10um sections at 90 degrees to the substrate, which I can use for IHC. > > Best wishes > Nick > > _______________________________________________ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > > > -- -- ********************************************** Geoff McAuliffe, Ph.D. Neuroscience and Cell Biology Robert Wood Johnson Medical School 675 Hoes Lane, Piscataway, NJ 08854 voice: (732)-235-4583 mcaul...@umdnj.edu ********************************************** _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail. _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet