[Histonet] IF on cell lines

Jeff Oliver jeff.oliver <@t> asterand.com
Mon Feb 16 09:12:35 CST 2009

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!

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Greg
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 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. 
is growing them on cell culture treated chamber slides and fixing with
formaldehyde.  Her problem is that after fixation and a few washes all
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 

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