Dear Craig,

You may find that you are getting differential fading of PI, particularly when you increase the light intensity. This may result in fading of the cytoplasmic fluorescence in preference to the nuclear labelling.

I have observed a similar phenomena where a relatively high background staining with particular samples diminished with time of exposure to light (without much loss of nuclear staining). This was using EthBr, which has somewhat different staining characteristics compared to PI - but it sounds like you have a similar observation with PI.

I have also noted that PI bound to very tightly packed DNA (for example in condensed chromosomes) is less susceptible to fading compared to DNA in a less condensed form.

regards, Alan.

BIOCON, specialists in confocal microscopy
7 Walhalla Drive, Ringwood East VIC 3135 Australia      phone: 61 3 9876 9822
Dr. Alan R. Hibbs


-----Original Message-----
From:   Craig Daly [SMTP:[log in to unmask]]
Sent:   Thursday, August 03, 2000 12:10 AM
To:     [log in to unmask]
Subject:        Propidium Iodide - odd results

Hi Folks,

Sorry if I have missed/forgotten some past discussion on this.  We have a
number of experiments now that show (in fixed blood vessels) a good nuclear
staining from PI when visualised at ex 364nm: em 400nm.  The 515nm line of
our UV laser gives the expected result (i.e. nuclear stain with light
cytoplasmic (RNA?) staining).  However, switching to UV and pumping the
laser up to 80-90% gives a nice clean image of the nuclei.  This PI is a
new batch from SIGMA and it is the first time we have noticed this effect.

I am puzzled by this.  We normally use H33342 and immediately suspected
some contamination but this has now been eliminated from our
investigations.  We have tried fluorescent beads to check the filters/lines
- everything ok there.  My last option is to consult the list.....any
suggestions gratefully received.

regards.

Craig.
_______________________________________________
Dr. Craig J Daly,
Research Fellow,
Wolfson Building (room 448),
West Medical Building (Lab 440),
University of Glasgow,
Glasgow G12 8QQ
Scotland.

Tel. 0141 330 3920
Fax. 0141 330 2923

Web sites:
www.cardiovascular.org
www.adrenoceptor.com
www.vascan.net
________________________________________________