[Histonet] Immunofluorescence question

[Gayle Callis]

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
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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)