[Histonet] neurons

[Valeria Berno]

Hi all,
 
I am new in the list and I already found it very useful so I hope to solve
this issue as well even if maybe is not "histology"related.....
 
I am working on primary neuronal cell culture trying to stain a membrane
receptor-GFP (with an anti-GFP antibody) and the MAPK activation.
 
Problems:
 
1- in the same coverslip if the antiGFP works...the anti MAPK doesn't!(and
viceversa). They are Mouse and Rabbit respectively and I am incubating them
together (milk 5% in TBS-T). 
 
2- Where I have the membrane receptor working however I would like to see
the internalization: I am fixing 15min in PFA4% on ice and permeabilizing
30min 0.2%triton But I don't get a good idea of the localization. Is that ok
as protocol or it is too strong?
 
Thanks in advance for all your suggestions or references
 
Valeria
 
Berno Valeria, PhD
 

EMBL Monterotondo Outstation

via Ramarini 32

00015 Monterotondo Scalo (RM)

Italy

Tel:  +39 06 90091 287

Fax: +39 06 90091 272

HYPERLINK "mailto:valeria.berno <@t> embl.it"valeria.berno <@t> embl.it

 
 

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