[Histonet] Immunofluorescence question

[Jimmy Lao]

We use PBS powder too, making 10x by kitchin, when we
do Immunofluorescence, we add Triton to 0.25%
please visit this website for protocol:
http://saturn.med.nyu.edu/research/dg/joynerlab/protocols.html
On my hand,  the protocol works well on a-GFP, but
background on a-Bgal, the worse the younger mouse
embryos like E9.5,  and I have a feeling that the 2nd
Ab step in 4 C overnight looks better.
I do want to see some suggestions.
Thanks!
Jimmy

--- Joanne Mauger <mauger <@t> email.chop.edu> wrote:

> Helllo All,
> 
> Do you find it necessary to use PBS vs TBS for
> fluorescence? My co
> worker recently switched from Dako TBS (addidng
> tween) to Labvision TBS
> which has tween in it. We thought these 2 TBS's
> would be equal, but her
> fluorescence did not work well, and there was lots
> of background. We
> switched her to PBS and all is well. 
> What I'm wondering- is there something different in
> the Labvision TBS
> that could effect our regular Immunos? So far we
> don't see any problems,
> and maybe hers was a fluke.
> Any thoughts?
> 
> Thanks,
> Jo
> 
> >>> Gayle Callis <gcallis <@t> montana.edu> 07/20/06 6:11
> PM >>>
> We purchase our Dulbeccos PBS in bulk, a dry powder
> that comes in
> quantity 
> to make 50 liters, goes into solution rapidly, from
> Sigma.  Do not buy
> the 
> DPBS with Mg or Ca added.   Weigh out 10 grams into
> 1 liter of
> distilled 
> water, pH is 7.4, works like a charm and very
> economical.   You can
> make up 
> to 5 or 10 liters at a time, so you have a slightly
> greater volume of 
> buffer sitting around, it is stable.  Or keep a 10X
> solution on hand so
> you 
> can dilute it when needed, volume to volume, much
> faster than weighing
> and 
> waiting.
> 
> How do you fix your FS for IHC work? Time in
> fixative?  Put your just
> cut 
> FS in front of a small fan (you can try different
> times, i.e. 15 min
> versus 
> 30 min, and use PLUS CHARGE slides, to improve
> drying. Losing a needed
> 
> section is certainly frustrating.  Heat application
> may destroy
> antigenic 
> epitopes.
> 
> 
> 
> At 12:44 PM 7/20/2006, you wrote:
> >Hi. Just have a few questions for those of you that
> do
> immunofluorescence.
> >Typically I do mostly skin punches and shaves for
> our dermatopath. 
> >My  question
> >is, do any of you order the PBS that is used for
> the procedure or make
>  it as
> >needed? I was making it up as needed, but there has
> been an increase
> in  the
> >cases and I wondered if anyone ordered
> commercially. If so, please let
> me
> >know a good vendor and I will get some ordered.
> Nothing worse that 
> >starting to
> >process your specimen just to notice that someone
> left you an empty
> bottle 
> >of
> >PBS. I've only been doing this procedure for a few
> months and I am
> always
> >looking for little tips to improve my technique. So
> far things have
> been 
> >going
> >well. I had a problem with tissue washing off
> before applying the
> antibodies
> >but  seem to have improved that area. I have tried
> applying heat,
> extending
> >drying  times etc., but inevitably there is still
> some tissue loss. I
> put 
> >several
> >sections on each slide with the control and seem to
> lose usually 1
> section of
> >  the patient tissue. I would love to have all
> sections stay until
> the
> >coverslipping stage but maybe that's my OCD and not
> realistic.
> >
> >Thanks for any info or words of wisdom you can
> share with me.
> >
> >
> >
> >Jodi L.  Putnam
> >HT (ASCP)
> >St. Thomas Hospital
> >Nashville,  Tennessee
> >615-389-9930
> >_______________________________________________
> >Histonet mailing list
> >Histonet <@t> lists.utsouthwestern.edu 
>
>http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> 
> 
> Gayle Callis
> MT,HT,HTL(ASCP)
> Research Histopathology Supervisor
> Veterinary Molecular Biology
> Montana State University - Bozeman
> PO Box 173610
> Bozeman MT 59717-3610
> 406 994-6367
> 406 994-4303 (FAX)
> 
> 
> 
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