Dear Confocalists:

A series of questions from a beginner interested in evaluating immunostained,
thick sections (50-100 µm) of formalin-fixed mouse brain.  I would like to
compare neurons labeled for tyrosine hydroxylase (TH) with neurons that do not
stain for TH.  Ultimately I would like to apply the "optical dissector" 
Gundersen et al, APMIS 96:857-881, 1988 and Coggeshall, TINS, 15:9-13, 1992)
for stereologic evaluation of individual neuronal (nuclear) volumes and surface
areas.

1) Preliminary sections of formalin-fixed brain appear to be significantly
autofluorescent. Is this the usual experience and will this 'background'
autofluorescence of negligible significance when compared with fluorescently
labeled or stained structures? 

2) Is there a good method for suppressing this background fluorescence?

3) Is there a good combination of stain and label that would allow me to
compare TH positive neurons and TH negative? In other words, is there a good
fluorescent neuron stain that I might use at one wavelength while
immuno-labeling with a fluorochrome that emits at another wavelength? 

4) And finally, is there a dependable method of calibrating optical sections as
one steps through the  pecimen in the Z axis? I have heard a great deal about
fluorescent particles of known dimension and I wonder if this is the solution.

	 Thank You in advance for your comments and advice. 

 	               Regards,
 	                       Ken Baker
    	                           Parke-Davis Research,
    	                                    Mississauga, Ontario,
    	                                            Canada,  L7J-2L8
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