[Histonet] Bone, fluorescent background, TRAP staining - triple info

[Gayle Callis]

You need to access this article in J Histochemistry Cytochemistry titled 
Histological Analysis of GFP expression in murine bone, JHC 
53(5):593-602,2005.  Although GFP is used, they dealt with autofluorescence 
very nicely using dual band pass filters on their microscope.   This is an 
elegant paper, one of the finest I've seen on fluorescence with GFP along 
with other staining methods.

Autofluorescence is a problem for those working with either double 
immunofluorescent staining or with GFP work.

If you use formalin or paraformaldehyde fixation, you NEVER get rid of 
autofluorescence, these fixatives usually increase autofluorescence 
levels.  For this very reason our work on bone or tissues attached to bone 
are done on undecalcified bone frozen sections, using the Cryojane tape 
transfer device.   Washing in general, does not decrease autofluorescence.

I read your previous message and reply from Chris van der Loos. You need to 
use two different species hosted secondaries as one way to reduce 
nonspecific binding and background fluorescence to avoid nonspecific 
binding background fluorescence, this is NOT autofluorescence - that means 
it is in the tissue before you ever start - and is the result of tissue 
components or induced by formalin or paraformaldehyde fixatives.   Both are 
problems, with autofluorescence either not easy to remove or 
impossible!!  Nonspecific binding by antibodies to tissues or other 
proteins introduced in your staining method is another story including 
dilutions, blocking, choice of secondaries, etc and is avoidable by how you 
chose to set up your double IFA staining.

With our double fluorescent staining we have done the following, as examples:

1. Goat antiGFP
2. Donkey antiGoat-FITC
3. Armenian hamster anti dendritic cell-biotinylated
4. Strepavidin-Alexa 555

Or one could do two rat antimouse primaries:

1.  Normal serum block is cocktail of goat and donkey serums,
2.  Rat antiMouse CD marker
3.  Donkey antiRat-FITC
4.  Rat serum block to ensure the next secondary does NOT detect the rat again
5.  Rat antiMouse
6.  Goat antiRat-RRX (rhodamine)

Please remember we do NOT use any aldehyde fixed bone samples.

Not sure of what you are doing exactly in your staining method, you only 
talked about secondaries.



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 (lab with voice mail)
406 994-4303 (FAX)