2008 – San Diego, California

Introduction to the seminar.
Co-chairmen:     Dr Richard W. Dapson
Vice President, Biological Stain Commission
Dr Richard W. Horobin
Associate Editor, Biotechnic & Histochemistry

Staining of macromolecules: possible mechanisms, examples, and strategies for investigation
Dr Poul Prento
Zoological Institutes, University of Copenhagen, Denmark (retired)

Staining a tissue section from an aqueous dye solution is a 2-step process. In Step 1, ion exchange builds up the concentration of appropriately charged dye ions in the immediate neighbourhood of the charged macromolecule. In Step 2, the actual intermolecular binding process occurs, where bonds, predominantly non-polar, are formed between macromolecule and dye. Step 2 follows the same principle as the processes which establish the tertiary structures of most proteins and nucleic acids. In this step the solvent (water) participates actively in the dye-binding process, which may be termed hydrophobic interaction and occurs concomitant with the formation of ordered systems of intramolecular hydrogen bonds. As fixation and processing do not lead to extensive protein unfolding, hydrogen bonding plays a minor or no role in the staining of most globular proteins, but can be important when staining extended protein structures such as collagens, elastin and amyloid. Staining of mucins and connective tissue glucosaminoglycans may also be by ion exchange followed by hydrophobic interactions as demonstrated by the phenomenon of metachromasia. However, if the dye ion is highly charged, only Step 1 (ion exchange) may happen. The latter is exemplified by staining with Alcian blue and possibly also by staining with colloidal iron. This attempt to explain staining from “the macromolecular point of view” will be further elaborated and exemplified, and some of the experimental strategies used in the investigations will be discussed.

Characterization of hematoxylin-based stain for use in the ThinPrep Imaging System
Wendy Stanick, Matthew McCormack, Steven Guertin, Kellie Forrestall and Michael Hopkins
Hologic Inc.

The ThinPrep® Imaging System is a technique of staining and imaging human cervical cells to aid in the diagnosis of cervical cancer and its precursors. The system utilizes a hematoxylin-based stain (Nuclear Stain) designed to be approximately stoichiometric. It stains the nucleus in contrast with the cytoplasm providing the ability to identify potentially abnormal cells for subsequent CT review. The success of this system depends upon consistent and reproducible slide preparation and staining. Meeting these strict requirements dictated that we investigate various measurements and analytical tests such as UV, HPLC and LC/MS to further characterize ThinPrep Nuclear Stain. These experiments have improved our ability to predict the stain’s functional performance over time. Additionally, we have investigated the effect of stain age, specimen quality and storage conditions.

Historic dyes and how to identify them
Chris  Cooksey  MSc CChem MRSC
Watford, England, UK

This presentation will cover the identification of historic dyes and pigments on ancient and not so ancient artefacts using a variety of physical and chemical techniques. Up to the mid-20th century, analytical tools were too blunt to resolve many of the identification problems. Since then advances in chromatographic and spectroscopic methods have yielded a wealth of data about the identity of colorants. This allows us, for example, to determine which species of madder (alizarin and purpurin) was used in dyeing and consequently, sometimes, to determine the geographic origin of the plant. In the same way cochineal (carmine) from Polish insects can be distinguished from that of American origin.

Roundtable discussion: Modern challenges with dyes and staining.
Drs Dapson and Horobin, Moderators.

The hematoxylin shortage of 2008 (John Kiernan, BSC Trustee)

Reports of BSC committees

Project and research reports from members

Informal open discussion of topics of interest