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