Leigh Ackland wrote:
> =
 
> Hello confocal users,
> =
 
> I was wondering if anyone has suggestions for getting rid of
> nonspecific/background fluorescence.  When I treat cultured breast
> carcinoma cells with pre-immune sera or a purchased non-immune serum, f=
or
> controls, using dilutions from 1 in 1000 to 1 in 4000, I get a great de=
al
> of non-specific labelling throughout the cells.  This is with DTAF as t=
he
> second antibody (usually anti-rabbit).  It makes it very difficult to w=
ork
> out the specific signal after treatment with, for example milk protein
> antibodies.
> =
 
> Generally I rinse cells in PBS for a few minutes, fix in 4% paraformald=
ehye
> for 15 mins, permeablise in 0.1% Triton X-100 and block with 1% bovine
> serum albumin in PBS (blocker) (for up to 18 hours),then add first anti=
body
> for 2 hours, wash 3 X over 20 mins in blocking buffer and add the DTAF =
(1
> in 200 in blocker) for two hours.  Use an Olympus AX with PlanApo 60X o=
il
> objective.
> =
 
> I would be very grateful if anyone could point out anything I can do to=
 get
> rid of the high background, or else maybe suggest another blocker solut=
ion
> that might be better.
> =
 
> Cheers,
> =
 
> Leigh Ackland
> Biological and Chemical Sciences
> Rusden Campus
> Deakin University
> Australia
 
Dear Dr Ackland,
 
To me your processing times seem to long. I suggest 2 min fixation in
fresh paraformaldehyde, wash for 2 x 10 min in PBS, mix Triton X and
blocking solution and incubate for 15 min, incubate for 1 hour with the
first antibody, wash 2 x 10 min, incubate with the DTAF for 30-60 min,
wash and mount. Good luck!
 
Greetings, G. Ocklind
-- =
 
Name: G=F6ran Ocklind, Ph.D.
Address: Uppsala Univ, Pharmacy /Div  Pharmaceutics
Box 580, 751 23 Uppsala, Sweden
http://pharm.bmc.uu.se/~galenisk
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