[Histonet] RE: Cell Culture Staining

[C.M. van der Loos]

Hi Kevin,
There is a method described by Harold Kerstens to embed your cells into 
paraffin blocks. Spin the cells down in an Eppendorf tube, remove the 
supernatant and mix the cells gently with 1 ml warm (65C) 2% agarose 
solution. Centrifuge again to concentrate the cells. Embed in paraffin 
as usally.
See paper by: Kerstens et al. JHC 48:709-718, 2000.
For the next time try to get some collagen sponges from some cell 
culturing company. Spin the (unfixed!) cells down and wash 3x with PBS 
(remove proteins). Discard supernatant and resuspend cells in a small 
volume. Insert sponge and allow the cells to adhere to the collagen (15 
min, RT). Fix sponge in buffered formalin and embed as usual.
Good luck!

Chris van der Loos
Dept. of Cardiovascular Pathology
Academical Medical Center
Amsterdam - The Netherlands

>-----Original Message-----
>From: Johnson, Kevin [mailto:KJohnson <@t> med.miami.edu]
>Sent: Friday, October 03, 2003 3:29 PM
>To: 'histonet <@t> lists.utsouthwestern.edu'
>Subject: [Histonet] Cell Culture Staining
>
>Greetings, all.
>
>A researcher just brought me (at Miller Time on a Friday!) two culture
>flasks of terminally differentiated Sertoli cells and requested H&E
>staining.  Well.  
>Conventional H&E obviously is out, since polystyrene is not compatible 
>with xylene.  Further restrictions: the cells cannot be scraped off, 
>they cannot be replated on glass coverslips, and for reasons of her 
>own, they cannot go back into the incubator pending an answer.  They 
>now have been fixed in situ with 10% neutral buffered formalin.
>
>Does anyone have a protocol for staining these puppies?  If not H&E 
>per se, then is there another staining method that might satisfy her?
>
>Thanks in advance, 
>
>Kevin Johnson
>University of Miami
>Diabetes Research Institute