10/3/96

Thanks for the responses. But it is apparent that I was not clear in

stating my problem. So I will give it another try.

I am trying to dissect out some very small <50 um blood vessels. The

main problem is that I cannot see them once the blood has washed out. I

would like to stain the vessels with something that is compatible with

immunohistochemical procedures using confocal microscopy. I am

interested in immunostaining for the innervation and then determining,

among other things, length density. Since most of the nerve are very

small I would like to image them using the confocal microscopy.

However, I am concerned that the vessel staining will interfere with the

fluorescence when viewed on the confocal either by scattering or

autofluorescence. Since alkaline phosphatase is present in most vessels

and is still active after paraformaldehyde fixation, this should would

work for the vessel stain. I am using rats and most likely the method

of Mayahara et al., Histochemie 11:88;1967. I hope that this has

clarified my problem and once again, any advice would be appreciated.

Michael J. Lyon

lyonm@vax.cs.hscsyr.edu

If you are fixing by perfusion,2% Evans blue in the fixative stains the vessels

without any conflict with horseradish peroxidase or for that matter the

fixation. I cannot comment on its fluorescence.

Kate Connolly

Katherine.S.Connolly@Dartmouth.EDU

Hello,

The Evans blue will emit very strong fluoresence in red but not green.

We use it as a nice counterstain when using FITC as our primary tag.

Bob

morphology core

dermatology

Univ. of Wash.

Seattle

underwoo@u.washington.edu

Mike -

You might wish to consider immunogold-silver staining (IGSS). There is no

interference by endogenous enzyme activity nor need to use fluorescence.

The signal is sensitive and permanent.

Feel free to consult our website for introductory protocols on the use of

colloidal gold at either the EM or LM level. One very nice reference is the

September, 1993 (Volume 16, Issue 3) edition of The Journal of

Histotechnology which was devoted to IGSS.

Regards, Don Cox

Donald P. Cox, Ph.D. GOLDMARK BIOLOGICALS

437 Lock Street Phillipsburg, NJ 08865

Telephone: (908) 859-2631 Fax: (908) 859-2875

E-Mail: goldmrkr@pop.fast.net or goldmarker@aol.com

Web Site: http://members.aol.com/goldmarker

"Goldmarking...is fun...and permanent, too!!

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