I have a lot of experience with collagen type labeling and one of the big
problems is that these elements can be highly charged and bind to anything
and everything, blocking especially with BSA included can add to this problem
setting the tissue up for non-speicific binding of ihc reagents.  For that
reason I have stopped using BSA in my serum block and now use animal specific
serum instead.
Patsy Ruegg
IHCtech
pruegg@colobio.com

Kathleen Spencer wrote:

> Hello Gail,
>
> Your message came in just as I was about to send the same request. I am
> having the same problem and nothing I have tried has worked either. I am
> at my wits end.
> I am using several different antibodies and 4 different alexa fluors,
> rabbit and mouse, all on rat brain sections. I ran a no primary control,
> a no secondary control and a section with both and they all three looked
> the same! And it was punctate, it looked so real!
> I took a section that had only been it PBS and saw nothing.
> If what you say is true about the blocking serum, you have made my day.
> We never thought of that.
> I will have to start over and redo three weeks of work, but at least it
> will be accurate.
> Hopefully someone out there can help us. I think most of them are at NSH
> so you may need to send this out again.
>
> Kathleen
> On Tuesday, October 1, 2002, at 11:39 AM, Gail Donegan wrote:
>
> > Hello All,
> >
> > I am staining for human extracellular matrix proteins using mouse
> > anti-collagen/fibronectin/elastin etc. I am using a
> > Fluorscein-conjugated
> > goat anti-mouse secondary antibody. Despite various preventative
> > methods I
> > am getting perfect staining on my negative (or null) control which has
> > no
> > primary added.
> >
> > I've used various dilutions, Bovine, human and rabbit serum block, and
> > multiple blocking steps
> >
> > all with no improvement. If anything, the secondary has greater affinity
> > for matrix proteins than it does for the IgG part of the primary. I'm
> > wondering whether, since the serum itself has matrix proteins in it,
> > whether to completely forego the blocking step.
> >
> >
> > Any thoughts or ideas extremely welcome,
> >
> > Thanks
> >
> > Gail
> >
> >
> >
> > Gail Donegan
> > Department of Clinical Engineering,
> > The University of Liverpool,
> > Duncan Building,
> > Daulby Street,
> > Liverpool,
> > L69 3GA.
> > U.K.
> >
> > Tel: +44(0)151 706 5198
> > Fax: +44(0)151 706 5803
> >
> > .
> >
> > Gail Donegan
> > Department of Clinical Engineering,
> > The University of Liverpool,
> > Duncan Building,
> > Daulby Street,
> > Liverpool,
> > L69 3GA.
> > U.K.
> >
> > Tel:    +44(0)151 706 5198
> > Fax:    +44(0)151 706 5803
> >
> >