Abcam Ad

Author Topic: Strange Pattern with DAPI  (Read 11336 times)

0 Members and 1 Guest are viewing this topic.

Offline Drake

  • Newbie
  • *
  • Posts: 1
Strange Pattern with DAPI
« on: April 18, 2008, 05:26:24 AM »
Hi everybody,

I need some advise about a strange pattern I obtained on yeast cells with a DAPI staining
The protocol was the following

On L-polylysine coated wells, the cells were air dried, fixated 30 minutes in 3,7 % formaldehyde, washed three time with PBS, fixated 5 min with methanol-ice cold and 30 sec with acetone ice-cold (until dried)
Then came the staining with my antibodies and final staining with DAPI

One of my slide then showed a diffusing DAPI pattern (whereas all control slides showed a typical nuclei restricted pattern)
given the pattern either it is a diffusion of nucleic acid within the whole cytoplasma of my cells (however a retrocontrol culture on the same showed that they are fully alive) or a staining of something else (either a cytoplamsic or a fungal cell component)
My question is then : does the DAPI is known to stain other components (sugar links for example if we assume that my labelling concerns the fungal cell) that nucleic acids molecules ?

here is a picture of the "problem"




Strange Pattern with DAPI
« on: April 18, 2008, 05:26:24 AM »

Offline gabor

  • SuperTech
  • ****
  • Posts: 23
Re: Strange Pattern with DAPI
« Reply #1 on: May 09, 2008, 08:01:19 AM »
looking your photo  it seems like DAPI stains the membrane and a point in the middle for each cells.
If I did not know they are yeast cells I could say they seem/are  nuclei of...
the membrane is very blushing.. and... if they are cell .. I can't see a lot of nucleic acids in the cytoplasm..
normally when the nuclei burst you can see many little fragments spreading in

what kind of yeast is it??
why do you fix for three times??


Offline excalibur

  • Global Moderator
  • GoldMember
  • *****
  • Posts: 1109
    • http://www.excaliburpathology.com
Re: Strange Pattern with DAPI
« Reply #2 on: May 09, 2008, 05:22:27 PM »
My question also is why are you fixing 3 times?

You should use formalin or methanol or acetone, not all 3.
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline monia

  • Newbie
  • *
  • Posts: 3
Re: Strange Pattern with DAPI
« Reply #3 on: February 28, 2009, 01:10:18 PM »
DAPI is a popular nuclear counterstain for use in multicolor fluorescent
techniques. ... Staining Pattern: Nuclei will be stained bright blue. ...
Milestones & Dedications, Strange Science Facts, Images & Movies, Grants

Offline ImmunoNYC

  • Global Moderator
  • GoldMember
  • *****
  • Posts: 684
Re: Strange Pattern with DAPI
« Reply #4 on: March 06, 2009, 11:46:21 PM »
I second excalibur's statement above.

Re: Strange Pattern with DAPI
« Reply #4 on: March 06, 2009, 11:46:21 PM »