[Histonet] Re: Immunofluorescence question

[Johnson, Teri]

Ditto the recommendation on making up a 10X solution of buffer, and
diluting liters or gallons worth as working buffer.

Unless I'm mistaken, I believe the immunofluorescent staining she is
doing are the direct IF markers on skin biopsies (IgG, IgA, IgM, C3,
maybe fibrinogen).

In my clinical days, we routinely did those using PBS buffer as a
diluent, and used PBS as a wash buffer. We did not use any detergent. As
these samples are not fixed (they are sent in Zeus/Michel's transport
medium), I would be concerned about the detergent washing away the
target before the antibody can label it. If you  have extra material and
good positive control, try using PBS and TBS with and without tween and
see if it makes a difference.

As for keeping the section on the glass, we always used positive-charged
slides. What micron thickness are your samples? And how long do you let
them air-dry prior to doing your immunofluorescent staining? Thicker
samples that have not adequately dried onto the slide might be part of
the reason your samples are coming off the glass as well.

Hope this helps!

Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110