Section Thickness
HPI obtains part of its precision by being insensitive to many sources of variability, such as variations in section thickness. Control of section thickness is important in obtaining precise registrations, but is not required for a good HPI signal. Post embedding labeling methods are two-phase mechanisms: IgGs partition from the fluid phase onto an essentially impenetrable surface. Even when samples are fully deplasticized, the density of cross linking in glutaraldehyde fixed material is so high that large radius of gyration molecules such as IgGs cannot penetrate. IgG binding and detection by HPI ^tm is thus independent of section thickness over a large range (certainly over 40-1000 nm). The practical import of this is not that one may relax the need for precision, but rather that very thin sections can be used without loss of signal strength, allowing probing of many signals in a single set of cells. Sections as thin as 40 nm have been used in HPI ^tm for optical microscopy (Marc, R.E., R. Murry and S.F. Basinger 1995 Pattern recognition of amino acid signatures in retinal neurons J. Neurosci. 15: 5106-5129.

Sectioning tips
As noted above, section thickness is has no little meaningful impact on signal strength in HPI ^tm. In practice, 200-250 nm sections are as easy to handle as routine sections >500 nm and yield substantial improvements in spatial resolution and quality of image registration. Serial sections should be cut but precision trimming to produce a ribbon is actually somewhat detrimental as HPI ^tmrequires the sections to be separated. Thus is it quite satisfactory to face blocs by hand rather than by glass knives. Each section should be placed on a droplet of filtered deionized water on a well of a multiwell glass slide, such as a 12 well Teflon® coated slide. One useful technique places the cells in a reverse N pattern, i.e. number the spots across the top as 1-3-5-7-9-11 and the bottom as 2-4-6-8-10-12 and place the sections down in sequence. This facilitates the placing of IgG droplets in various patterns. If you wish to probe every other section with the same IgG, then simply placing the IgG on every well of the upper or lower row will suffice. If you wish to use electron microscopic thinness sections such as silver or gold or even superthin sections, it is easiest to remove each section from the boat by floating it in a droplet within a transfer loop. Transferring the section to the slide requires care and a stereomicroscope or magnifying glasses. Gently touch the leading edge of the loop to the slide at about 30 degrees from horizontal and flex the loop with a little downward pressure to force the droplet to touch the glass. Wick the water away slowly with a small paper point (available from Ted Pella, Inc.) placed just outside the front edge of the loop and pushed into the crease between loop and glass. The section should slowly descend to the glass surface and, with luck, should remain free of folds. While tedious, use of 60 nm sections allows sampling 12 serial haptens in less than 3/4 um.

Deplasticizing
Deplasticizing epoxy resin sections involves basic attack on the end-linked epoxide rings. In electron microscopic post-embedding immunocytochemistry, strong aqueous hydrogen peroxide is used to disrupt the links but attacks proteins as well. The etching is quite limited however. For light microscopic purposes, the entire epoxy resin matrix may be removed by using anhydrous bases such as sodium ethoxide or methoxide These agents will disrupt the end-links and rapidly dissolve the polymer but, in the absence of water, will not attack most hydrophilic biological molecules. Indeed, the addition of even trace amounts of water to the solution will produce a stunning slide-cleaning solution that will remove all traces of tissue. On the whole, some proteins, peptides and many amino acids have been successfully deplasticized with these methods, restoring strong immunoreactivity.

Deosmication
Electron microscopic immunocytochemistry requires deosmication for the restoration of some immunoreactivity, the suppression of spurious gold deposition at osmium rich sites, and for light microscopic immunocytochemistry, the removal of osmium itself as it will spontaneously catalyze the silver intensification reaction and quench the gold-mediated reaction. It is not clear that osmium itself seriously impedes hapten immunoreactivity as reasonable immunogold GABA signals can be had in fully osmicated electron microscopic samples. However, osmium does seem to restrict hapten mobility which may attenuate signal strength significantly for some residues. Deosmication involves the reversal of the multiple coordination bonds formed by reoxidizing osmium with very reactive species such as periodates. Typically sodium metaperiodate or periodate are used after deplasticizing to reverse the osmication. For most small haptens, this seems fairly innocuous and full immunoreactivity of over 20 small haptens (mostly amino acids) can be restored. However, some molecules such as glutathione are strongly modified by osmication and recovery of immunoreactivity may require extensive periodate bleaching. Thus more complex species, and especially those containing thiols, are likely not always compatible with electron microscopic analysis if osmication is desired.


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