Hi,
    I think your 1:5000 was over stained. The staining you saw in the
negative and in the cytoplasm was probably endogenous biotin. Since the
intensity was good though and it faded in the 1:7000 I think the first is
better but you should try to eliminate the background staining by either a
biotin block or a non biotin secondary detection system (Envision or Mach II
or an alkaline phosphotase kit).

my $0.02,
Amos Brooks

----- Original Message -----
From: "Thu-Thao Pham" 
To: 
Cc: ; 
Sent: Thursday, November 29, 2001 1:52 PM
Subject: testing antibody dilution


> Dear Histonet,
>
> I'm trying to figure out the right dilution for my antibody stained in
human
> liver cell-line. I ran several dilution and along with a positive control.
> I'm expecting my antibody to be located in nucleus. Positive control
showed
> it's so and it also stained in cytoplasmid. On my human liver cell-line it
> showed no nucleus stain (good for me). However, there is very fainted
> stained on the cytoplasmid. Two dilution of primary antibody I don't know
> what I can conclude as my good dilution for my antibody when compare with
> the positive control.
>
> 1) I antibody at 1:5000 showed on a + control showed a complete staining
in
> nucleus and in cytoplasmid. However, negative human liver cell-line showed
a
> no nucleus staining (good for us), but very fainted here and there in the
> cytoplasmid.
> 2) I antibody at 1:7000 showed on a + control showed a complete staining
in
> nucleus (but not strong) and in cytoplasmid. However, negative human liver
> cell-line showed a no nucleus staining (good for us), as well as in the
> cytoplasmid.
>
> My question is, what would be my recommendation dilution for my antibody
> that I have tested after compared with my positive control. Should I go
for
> the 1:5000?
>
> Any opinion or advice would be more appreciated.
>
> Thao