>        I'm staining spinal cord and medulla's neurons from rats' fetuses,
>10 days old, with the following primaries antibodies: Rabbit anti Glycine
>1:2500, 1:4000; Rabbit anti GABA 1:100, 1:200; Rabbit anti GAD 1:300,
>1:500; and anti-synaptophysin 1:150, 1:300. I'm using FITC 1:100 or
>Rhodamine 1:100 as my secondary antibodies. I keep getting crossover
>between FITC and Rhodamine despite the different dilutions of the primary
>antibodies I have used. Does anyone has any suggestion?
>Thanks in advance
>
>Ciprian Almonte
>Research assistant
>Medical College of Pennsylvania
>Dept. of Anatomy and Neurobiology
>3200 Henry Ave.
>Philadelphia, PA 19129
>Voice: (215) 842-4081
 
 
Hi Ciprian,
 
The dilution that you listed for your secondary antibodies look pretty low
(1:100). Since I don't know the starting concentration of those antibodies,
I cannot really tell however. From Cappel, Inc., I generally get FITC- or
RITC-conjugated antibodies at approx. 10-20 mg/ml concentrations and I
begin my test dilutions at 1:500 (most concentrated) and seem to settle
with dilutions of 1:700 to 1:1000. So, for immunofluorescence, my stuff has
the least background if the secondary antibody concentration is below 2
micrograms/ml.
 
I would also try using different blocking reagents (BSA, milk, gelatin,
serum), because they will block more, or less, effectively dedending upon
your tissue and fixation process.
 
Hope that this is helpful,
 
Dave