[Histonet] culture on coverslips troubles

[Monfils, Paul]

It depends a lot on what kind of cells are being cultured. When cells are
grown in a culture flask, some kinds will come loose into suspension if you
just hit the flask with your hand, while other cells require trypsin or
other enzymatic treatment to get them loose. If you are dealing with a
culture that has poor adherence, the cells can be firmly attached to the
glass by using a coagulative fixative like Bouin's solution or alcoholic
formalin.  This works well if you are going to do a standard chemical stain
on the cells.  But for IHC it's a bit more complicated.  Coagulative
fixatives often denature antigens to the point where their antibodies no
longer recognize them. And fixatives commonly used for IHC, like acetone or
aqueous formalin, don't coagulate proteins enough to make the cells adhere.
Air drying the coverslips without fixation will cause the cells to stick
fast, but not without severe loss of morphology.  If your antibody will
tolerate formalin fixation, I would try brief (1 minute) immersion in 5%
alcoholic formalin (1 part formaldehyde to 19 parts 70% ethanol). If this
interferes with your antibody binding, try brief fixation in plain 70%
ethanol. In fixing cultures on coverslips, drain off the excess fluid from
the coverslips, and put them into the fixative when they are just "damp".

> ----------
> From: 	histonet-bounces <@t> lists.utsouthwestern.edu on behalf of
> LaCinda Burchell
> Sent: 	Tuesday, July 19, 2005 10:16 AM
> To: 	histonet <@t> pathology.swmed.edu
> Subject: 	[Histonet] culture on coverslips troubles
> 
> Greetings Histonetters:
>   I'm being asked to give advice about how to keep cultured cells on their
> cover slips during IHC for Confocal Microscopy.  Is there a specific
> technique for washing the cover slips without losing cells?  Should the
> cover slips be "air-dried" at some point?  What fixative is best for
> confocal microscopy?  Are there positively charged cover slips out there?
> Last question: Does anyone know of a list server for culture people?
> Marketing people please feel free to respond to me regarding these
> questions if you think you can be of service.   I thank you all, and I
> send thanks for my questioning friend.
> 
> LaCinda Burchell, BA, AS, HT(ASCP)
> University of Wisconsin-Madison, Medical School
> Asthma and Allergy Research IHC Lab
> 600 Highland Ave.  CSC  K4/913
> Madison, Wisconsin  53792
> 
> Phone: 608-262-3518
> FAX:     608-263-3746
> 
> 
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