2 replies to this topic

[pooashri]

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.

[doxorubicin]

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.

[Tabaluga]

pooashri, on 25 March 2012 - 02:01 AM, said:

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.