[Histonet] IF on cell lines

[Jeff Oliver]

Do you know if the slides are charged?  The adherent cells lines I've
worked with prefer a charged surface to stick to.  This was a problem a
few years back when I ordered the wrong petri dishes!  Like Greg, I grew
cells on the coverslips rather than the slides themselves.  3%
paraformaldehyde or cold methanol were my standard fixations.
Hope this helps!
-Jeff



-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Greg
Dobbin
Sent: Monday, February 16, 2009 9:22 AM
To: awatanabe <@t> tgen.org
Cc: Histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] IF on cell lines

Hi April,
This may not be useful to your colleague at all, but here goes: I used
to place a round glass sterile coverslip in the bottom of each well of a
24-well culture plate before seeding and then grew the cells on the
coverslip. When ready the coverslip was removed (using a hyperdermic
needle with the bevelled tip bent backwards to catch the edge of the
cs). The coverslips were then picked up with "eyelash" forceps and
placed in a little porcelain coverslip rack and placed in cold acetone
(-20 C) to fix for 10 mins. Following IF staining the round coverslip
was mounted on a regular coverslip which was in turn mounted on a glass
slide for viewing.

In case you haven't already figured this out, acetone melts the plastic
chamber slides. My coverslip method, while effective, is obviously much
more laborious (which I suppose is why the chamber slide was developed
in the first place! LOL). Hopefully the 4% paraform. works. Is methanol
fixation an option??
Greg

Greg Dobbin, R.T.
Chief Technologist, Anatomic Pathology
Dept. of Laboratory Medicine,
Queen Elizabeth Hospital,
P.O. Box 6600
Charlottetown, PE    C1A 8T5
Phone: (902) 894-2337
Fax: (902) 894-2385

"I find that the harder I work, the 
more luck I seem to have."
- Thomas Jefferson


>>> Aprill Watanabe <awatanabe <@t> tgen.org> 2/16/2009 9:13 AM >>>
I have a colleague who is trying to do IF on cell culture cell lines. 
She
is growing them on cell culture treated chamber slides and fixing with
4%
formaldehyde.  Her problem is that after fixation and a few washes all
the
cells are detaching.  Any suggestions?  I think she is going to try 4%
paraformaldehyde first as someone has already switching to that.

Aprill Watanabe, B.S.
Research Associate
Integrated Cancer Genomics Division
Tissue Microarray Center (TMA)
Translational Genomics Research Institute (TGen)
main: 602-343-8822
Fax: 602-343-8840
awatanabe <@t> tgen.org 
www.tgen.org 

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