Hello all, and especially John Kiernan who I hope will respond.

I have been trying to use the Winkelmann Schmitt procedure listed in
Kiernan's book. I will briefly describe the problem I am having below, but
my real question is about modifying the physical developer. I want to make
it less sensitive to how much sliver is carried over from the impregnating
solution. 

For those not familiar with the stain, it is a silver impregnation similar
to a Bielchowski (20% Ag) but with a different physical developer: 0.2%
hydroquinone and 1% sodium sulphite. In the Biel I am familiar with, the
developer is added to the silver solution. This stain is the reverse where
the working silver must be carried into the pristine developer. The stain
reveals axons in skin sections.

Before I mention any details, here's my question: does anyone have a rough
estimate of the optimal concentration of silver ions in the working physical
developer? For this or any other physical developer? I am going to
experiment with adding silver nitrate directly to the developer before use.
This is done in other procedures and is done in several photographic
techniques where physical developers came from. I'm not looking for a hand
out, just a little guidance so I don't start shooting in the dark. 

The problem I'm having is splotchy development of the siver. I've narrowed
it down to non-uniform carryover. John says in a note that the rinse before
the developer must not eliminate all silver since the carryover is the
source of silver ions for the developer's work. My problem is I can't seem
to get the magic timing and handling so I get enough and not too much. There
is some staining where the sections retain free silver more avidly. This
must be the result of rapid local development before the silver diffuses out
into the bulk of the developer solution. If I don't rinse enough before
going into the developer then there is enough local free silver to
immediately turn the section all black. There is a magic middle ground
somewhere that I can't find. I don't like stains that require "magic
touches".
 
I can see two avenues to experiment:

First, reduce the volume of developer used so that the effective carryover
concentration will be greater. My problem may be that I am unfamiliar with
free floating techniques. The procedure lists a formula for 100ml developer.
I have been using it all in one beaker. How much solution per section do
people usually use for thick free floaters?

Second, introduce a known concentration of silver ions directly into the
developer and rinse sections thoroughly to eliminate "hot spots" of
developer action. I like this second option because it seems easier to
control. But I have no idea where to start the concentration. Dilute I know.
And is there a best way to introduce the silver to the developer?

I have tried both de-fatted sections and non-de-fatted sections and get
pretty much the same results. Actually I think the non-de-fatted sections
carry over a little better probably because of the reduced permeability.

Just for the record, yes I am using 50-80 micron frozens of formalin fixed
skin. And yes I am using free floating techniques - I had to go down to
microbiology to make my little glass hockey sticks! No one else is allowed
bunsen burners. All part of the fun! And I am in the process of digging up
photography references to beter understand the variations of physical
developers - looking for clues. I have not pulled Winkellman's original
paper yet.

O.k. O.k. Sorry to run on! Thank You in advance for any info! And Thank You
John Kiernan for the excellent book!

-brice
Neuropathology
Brigham & Women's Hospital, Boston