p-Phenylenediamine
preservation is improved to the extent that specimens appear similar in contrast to those post-fixed with osmium tetroxide. Use of 1% PPD during the dehydration of cell cultures permits visualization of the cell cultures aſter osmication without the use of a dissecting microscope to locate areas of high cell density. Figures 1 and 2 show specimens that were fixed in buffered acrolein with 1% (wt/vol) tannic acid, dehydrated and infiltrated with 1% (wt/vol) PPD in all the ETOH containing steps and embedded in LR White followed by polymerization in gelatin capsules at 50–55 C. Improved immunolocalization has been accomplished using conventional bench methods for specimen processing and labeling as well as with cold microwave assisted protocols.
References [1] JM Ledingham and FO Simpson, Stain Technol 45(255) (1970).
[2] JM Ledingham and FO Simpson Stain Technol 47(239) (1972).
Figure 2: Higher magnification of localization of chemotaxis protein tsr in E. coli with 12-nm colloidal gold (arrows) labeled secondary antibodies. Scale bar equals 100 nm.
Results Sections of specimens embedded in epoxy resins require
oxidation with periodic acid in the immunolabeling protocol, whereas sections from specimens embedded in acrylic resins usually do not require oxidation. Overall, the antigenic preser- vation and immunolabeling efficiency is greatly improved by use of this protocol. In addition, the morphological
[3] J R Guyton and K F Klemp, J Histochem Cytochem 36(1319) (1988).
[4] AK Bal, Stain Technol 65(91) (1990). [5] KD Phend, A Rustioni, and RJ Weinberg, J Histochem Cytochem 43(293) (1995).
[6] S–H Brorson, I Halvorsen, L-C Lonning, G Slaattun, M Sletten, and S Rashid, Micron 30(561) (1999).
[7] EA Ellis, Microscopy Today 14(4) (2006) 32. [8] Appreciation is expressed to Dr. Rosemary McAndrew for use of the micrographs in Figures 1 and 2.
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