Glyoxal fixation: how it works and why it only occasionally needs antigen retrieval

Over the past 13 years, glyoxal has become the leading alternative to formaldehyde as a histological fixative because of its low inhalation risk, faster reaction rate and selective control over crosslinking. The latter attribute is especially important, because most of the difficulties relating to use of formaldehyde-fixed specimens for immunohistochemistry stem from its aggressive crosslinking behavior. With suitable catalysts or other reaction accelerators, glyoxal forms 2-carbon adducts with nearly all end groups in proteins and carbohydrates, leaving most of them unimpaired for subsequent immunohistochemical demonstration. Only arginine is seriously impaired by the formation of imidazoles, which is the basis for the well known arginine blockade method using glyoxal. A special glyoxal-specific antigen retrieval method using high pH and high temperature effectively reverses the blockade and restores immunoreactivity. Other methods for antigen retrieval are rarely beneficial and in most cases damage the specimen. Special stains work well, except silver methods for Helicobacter pylori. Routine hematoxylin and eosin preparations exhibit clarity and cellular detail rarely seen with formaldehyde.     

Accomplishments of the Trustees and laboratory staff of the Biological Stain Commission, 2002–2013

During the 12 years from 2002 to 2013, the Trustees and laboratory personnel of the Biological Stain Commission (BSC) can claim many accomplishments. These accomplishments are itemized under 11 categories: continuous publication of the official journal, Biotechnic & Histochemistry; production of four special issues of Biotechnic & Histochemistry devoted to specific dyes or stains; standardization of staining and dye purity; mechanisms of staining and prediction of dye behavior; publication of books or book chapters; effects of fixation and processing on staining; cancer research; immunohistochemistry; BSC Laboratory activities; miscellaneous publications; and administrative accomplishments.     

News from the Biological Stain Commission No. 15

In the 15^th issue of News from the Biological Stain Commission (BSC), under the heading of Regulatory affairs, the Biological Stain Commission’s International Affairs Committee presents information from the plenary meetings of the International Standards Organization ISO/TC 212 Clinical laboratory testing and in vitro diagnostic test systems held on August 22–24, 2012 in Berlin, Germany. An additional discussion of the use of food dyes in India also is included.     

Domain-dependent protein folding is indicated by the intracellular kinetics of disulfide bond formation of human chorionic gonadotropin beta subunit.

We have measured the intracellular rates of formation of the six disulfide bonds in the human chorionic gonadotropin beta subunit (hCG-beta) to determine whether the folding pathway of this molecule can be described by a simple sequential model. If such a model is correct, the formation of disulfide bonds, which is indicative of tertiary structural changes during protein folding, should occur in a discrete order. The individual rates of disulfide bridging were determined by identifying the extent of disulfide bond formation in hCG-beta intermediates purified from choriocarcinoma cells that had been metabolically labeled for 40 to 120 s and chased for 0 to 25 min. The results of these kinetic studies describe a folding pathway in which the disulfide bonds between cysteines 34-88, 38-57, 9-90 and 23-72 stabilize, in a discrete order, the putative domain(s) involving amino acids 1-90 of hCG-beta. However, the S-S bonds 93-100 and 26-110 begin to form before the complete formation of the disulfide bonds that stabilize the amino acid 1-90 domain(s), and continue to form after complete formation of these disulfide bonds, suggesting that hCG-beta does not fold by a simple sequential pathway. The order of completion of each of the six disulfide bonds of hCG-beta is: 34-88 (t1/2 = 1-2 min), 38-57 (t1/2 = 2-3 min), 9-90 and 23-72, 93-100, and 26-110. Moreover, 60-100% of each of the six disulfide bonds form posttranslationally, and nonnative disulfide bonds do not form in detectable amounts during intracellular folding of hCG-beta.     

What regulates secretion of non-stored proteins by eukaryotic cells?

Protein secretion is conventionally viewed as taking place by either of two cellular routes, a regulated pathway, involving external stimuli and secretory granules, and a presumptive ‘constitutive’ pathway, which does not involve hormonal or neuronal stimuli or the production of secretory granules. The evidence reviewed here strongly suggests that there are post-synthesis rate-limiting steps for many proteins released by the ‘constitutive’ pathway and, hence, that regulation in some sense is involved here too. The nature of these rate-limiting determinants and events is discussed.