[Histonet] Immunofluorescence question

[Gayle Callis]

We use Dulbeccos PBS and TBS interchangeably and with a small amount of 
Tween only just for sheeting action over sections.

We make up our own TBS, a 10X solution, and dilute it when needed to make a 
0.05M TBS, at final pH of  pH 7.6.  Have you checked the pH of your buffer 
to make sure there is not some change there? Check the MSDS, product sheet 
or call Lab Vision tech services to discuss their buffer.  I would bet 
their TBS is a standard recipe for making it up, and works well.   For IFA 
work, we use 0.025% Tween 20 and for IHC 0.05% Tween 20.

We never lose fluorescence (IFA) nor staining with enzyme 
immunohistochemistry with either buffer, and you may want to sleuth for 
another reason why the fluorescence was not optimal.  Background generally 
is not caused by buffer problems, but another reason, cross reaction, too 
high antibody concentration, fixation issues.

At 05:33 AM 7/21/2006, you 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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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)