Re: [Histonet] Immunofluorescence question

From:"Joanne Mauger"

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  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@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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