A quick-and-dirty way of creating positive controls for TUNEL staining is to
take some cells from cell culture and just induce apoptosis in them, either by
exposure to UV light, viral infection, or actinomycin treatment (the actual
method you use to induce apoptosis will vary with the cell type, as will the
duration of time you will need to wait to collect, spread, and fix the cells).
When the cells are at the stage at which you would see DNA laddering, then fix
the cells in paraformaldehyde, dry them onto treated slides, and then TUNEL
stain them (you can also permeabalize the membranes with a weak Triton-X
solution, although the drying process should be sufficient).

-Tom Clarke-

Mandy Annis wrote:

> Teri,
>
> The following instructions for creating positive control samples for
> apoptosis are from the Intergen Company-ApopTag In Situ Apoptosis Detection
> Kit :
>
> 1.  Pretreat section (they indicate any tissue will work) with DN Buffer
> (30mM Trizma base-pH 7.2, 4mM MgCl2, 0.1mM DTT) at room temp for 5 min.
>
> 2.  Dissolve DNAse I in DN Buffer to final concentration of 1.0-0.1 ug/mL,
> apply to sample and incubate 10 minutes at room temp.
>
> 3.  Rinse 5 times for 3 min. each with dH2O.
>
> I have no experience using this protocol, but hope it works well for you!
>
> Mandy
>
> Mandy,
>
> Yes, please do pass the information along.  The more I know about this, the
> better!
>
> Thanks,
>
> Teri
>
> -----Original Message-----
> From: Mandy Annis [mailto:mandy_annis@usgs.gov]
> Sent: Thursday, March 14, 2002 10:10 AM
> To: Johnson, Teri
> Subject: Re: control tissue for TUNEL
>
> Teri,
>
> My limited experience with apoptosis involved a kit from the Intergen
> Company which supplied a rodent mammary gland (3-5 days post weaning) as a
> positive control.  The instruction manual further suggests preparing a
> positive control from any tissue by treating with DNase.  Full instructions
> are supplied.  I would be happy to pass them on to you if this is of
> interest.
>
> Mandy
>
> **********************************************************************
> Mandy L. Annis
> Columbia Environmental Research Center
> 4200 New Haven RD  Columbia, MO  65201
>
> Phone:(573)-441-2940
> Fax:(573)-876-1896
> e-mail:mandy_annis@usgs.gov