P. Echeagaray
CAWAGN@MONSANTO.COM wrote:
>
> Fellow flowers:
>
> I am working with mouse hepatocytes from both CD-1 and p53 knockout
> mice. We are obtaining hepatocytes by collagenase perfusion followed
> by percoll gradient separation. After separation, I should have a
> pretty pure hepatocyte population. When I look at the cells on
> Forward vs. Side scatter, there appears to be approximately three
> separate populations. I am not sure if they are all hepatocytes that
> differ in size, it there are clumps of cells, or if there is
> contamination of other cell types in my cell prep.
>
> I need any information that any of you might have pertaining to
> markers that would be hepatocyte specific, and any
> fixation/permeabilization methods that might work.
>
> I tried to use anti-cathepsin D and anti-transferrin (rabbit
> polyclonal) from Dako, but didn't see any fluorescence above
> background. This may have been due to my method of fixation and
> permeabilization. I used 1% paraformaldehyde/20fg/ml lysophosphatidly
> choline (2 min. RTx), followed by absolute methanol (10 min. on ice),
> followed by 0.1% lysophosphatidly choline (5 min. on ice).
>
> For staining, I blocked with Caltag anti-FC receptor (MM7200) for 30
> min. at on ice, washed 2 x with 10% heat inactivated rabbit
> serum/Dulbecco's Phosphate Buffered Saline without Calcium,Magnesium,
> followed by 30 min. incubation on ice with a 1:20 dilution of 1x
> antibody. The antibodies are diluted in 10% heat inactivated rabbit
> serum/Dulbecco's Phosphate Buffered Saline without Calcium,Magnesium.
>
> After incubation, the cells are washed 2 x with the rabbit/DPBS and
> then blocked again with anti-FC receptor as above. The cells were
> then incubated with a 1:40 dilution of Santa Cruz goat anti-rabbit
> Fitc for 30 min. on ice.
>
> After incubation, the cells are washed 3x with rabbit/DPBS, and fixed
> in 1% paraformaldehyde until analysis.
>
> I am not sure if the antigens may have been washed out of the cells by
> my fixation method. I am also not completely sure if the antigens are
> cytoplasmic , or if they are nuclear.
>
> What I hope to do is identify the hepatocyte population fluorescently,
> and then backgate onto the FSC vs. SSC plot.
>
> Thanks to those of you who have already helped me a great deal and in
> advance to those who will,
>
> Connie Wagner
> Monsanto Environmental Health Laboratory
> 645 S. Newstead Ave.
> St. Louis, MO
> email address: cawagn@ccmail.monsanto.com
> phone: (314) 694-7971
> fax: (314) 694-7938