Colocalization - Fluorescence Histological Double Labeling (Nov/04/2004 )

I'm trying to colocalize Tyrosine Hydroxylase and the GDNF receptor (GFRa1). Tyrosine Hydroxylase labeling (Rhodamine conjugated secondary antibody) works OK but I'm having problems with FICT conjugated secondary antibodies as I get to much background and poor cell labeling. I've tryied to dilute secondary antibodies but with little labelling improvement. Hope someone can help with this, José W.

a) you can reduce your background by treating your samples with 1 mg/ml sodium borate (in PBS) for 10 min after you've permeabilised and before you incubate with primary.

Also, try to filter your antibodies through a 0.2 micron filters (micro filters for syringes are available). Both your primary and your secondary. This way you'll avoid big clumps on your samples.

good luck,...