Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Aberrant (apparently) nuclear immunostaining for VEGF in Glial cells

Immunocytochemistry Astrocytes cell culture VEGF

  • Please log in to reply
2 replies to this topic

#1 pooashri

pooashri

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 25 March 2012 - 02:01 AM

I recently stained my astrocyte culture derived from spinal cord of embryonic rats for VEGF. However, the staining repeatedly comes out to be prominently nuclear, and comparatively less cytoplasmic. I searched for literature, and could not find much related to nuclear localisation of VEGF in healthy glial cultures.


Could anybody present any explanation, or suggest any changes in the ICC protocol? we have used rabbit Anti VEGF (Abcam, 1:500) labelled with Anti rabbit CY3 (santacruz, 1:200), preceded by the antigen retieval SSC formamide treatment, and 3% BSA blocking. The cells were fixed with 4% PFA, at room temperature for 20 min.

PS: the same antibody showed a clear cytoplasmic staining with the NSC-34 cells (fixed with chilled Methanol, 4 min) stained in the same batch.

#2 doxorubicin

doxorubicin

    Veteran

  • Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 192 posts
15
Good

Posted 26 March 2012 - 01:52 PM

An immunostaining experiment is only as good as its negative control. If there is no way to use knockout or knockdown cells as a negative control, then you just have some signal that may or may not be dependent on your protein.

#3 Tabaluga

Tabaluga

    Making glass out of shards

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 363 posts
47
Excellent

Posted 02 April 2012 - 10:56 AM

I recently stained my astrocyte culture derived from spinal cord of embryonic rats for VEGF. However, the staining repeatedly comes out to be prominently nuclear, and comparatively less cytoplasmic. I searched for literature, and could not find much related to nuclear localisation of VEGF in healthy glial cultures.


Could anybody present any explanation, or suggest any changes in the ICC protocol? we have used rabbit Anti VEGF (Abcam, 1:500) labelled with Anti rabbit CY3 (santacruz, 1:200), preceded by the antigen retieval SSC formamide treatment, and 3% BSA blocking. The cells were fixed with 4% PFA, at room temperature for 20 min.

PS: the same antibody showed a clear cytoplasmic staining with the NSC-34 cells (fixed with chilled Methanol, 4 min) stained in the same batch.


You could do a western blot (with your AB and the respective cells) in order to find out whether your AB just recognizes VEGF (single band at the size of VEGF) or if it perhaps gives several bands, indicating that it recognizes other, possibly nuclear proteins too.

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 






Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.