The application of heated detergent dewaxing and rehydration to immunohistochemistry.

Hot commercial dishwashing detergent has been used to deparaffinize and hydrate formalin fixed, paraffin embedded sections for immunohistochemistry. Fifty-five antibodies, used routinely for diagnosis, were used to compare hot detergent dewaxing with the proprietary hydrocarbon-based dewaxing reagent supplied with the Bond Max immunohistochemistry system®. A 2% concentration of commercial dishwashing detergent in distilled water was heated to 90° C and paraffin sections were treated twice for 1 min each. Nearly all antibodies gave equivalent results except CD10 and CD57 (hydrocarbon-based dewaxing better) and CD45 and alpha fetoprotein (detergent dewaxing better); the differences, however, were minimal. There also was a significant cost saving using detergent dewaxing.

Tape transfer sectioning of tissue microarrays introduces nonspecific immunohistochemical staining artifacts.

Tissue microarrays place tens to hundreds of formalin fixed, paraffin embedded tissue cores into a paraffin block in a systematic grid pattern that permits their simultaneous evaluation in a single section. The fragmented nature of the tissue cores often makes sectioning of tissue microarrays difficult so that the resulting disks of tissue lose their shape, fracture or fall out of the paraffin section altogether. We have evaluated an alternative sectioning protocol for stabilizing the tissue microarray surface by placing an adhesive tape "window" over the face of the paraffin block prior to sectioning. Once sectioned, the tape/sections are transferred directly onto coated microscope slides, thereby avoiding routine floating of sections on a water bath. After sectioning with either the tape transfer or standard protocols, slides were stained either using hematoxylin and eosin or immunohistochemistry using antibodies to S-100 protein and the tissue specific antigens, keratin (AE1/3) and the leukocyte common antigen CD45. We found that the tape method produced thicker sections that were darker and more densely packed with loss of tissue definition compared to sections prepared using water bath flotation. Quantitative image analysis of immunohistochemical staining demonstrated that the tape method produced a higher incidence of nonspecific staining, which raised the potential for false positive staining.

Current applications of orcein in histochemistry. A brief review with some new observations concerning influence of dye batch variation and aging of dye solutions on staining.

Current uses of orcein to demonstrate elastic fibers and, following permanganate oxidation (Shikata's modification), hepatitis B surface antigen, copper associated protein, and sulfated mucins, are reviewed. Variations in staining performance with batch of dye and age of dye solution is also discussed. Additional experimental findings support the view that the orcein stain for elastic tissue and Shikata's modification produces consistent, high quality results as long as appropriate controls and suitable dye batches, e.g., Biological Stain Commission certified dyes, are used.

Improved demonstration of mast cells using alcian blue tetrakis (methylpyridium) chloride.

Many batches of alcian blue dye are incompletely soluble at the low pH used for demonstrating mast cells. An improved technique using alcian blue tetrakis (methylpyridium) chloride (alcian blue pyridine variant) is described here. It produces stronger mast cell staining than other alcian blue stains tested.

Bromine-, chlorine-, and mixed halogen-substituted 4-methyl-2(5H)-furanones: synthesis and mutagenic effects of halogen and hydroxyl group replacements.

The versatility of 4-(hydroxymethyl)-2(5H)-furanone as a starting point for the synthesis of several bromine and mixed halogen analogues of the potent water mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) has been demonstrated. However, in some preparations the yields of desired products were lower for bromine- than chlorine-substituted counterparts. A total of 12 bromine-, chlorine-, and mixed halogen-substituted 4-methyl-2(5H)-furanones were tested repeatedly in 10 independent experiments for levels of Salmonella typhimurium (TA100) mutagenicities. The purpose of these experiments was to determine the mutagenic response to changing halogen content, type, and position as well as to learn the measure of these responses in the presence and absence of the C-5 OH group. Mutagenicities reached levels of 10(3) and 10(2) rev/nmol for all trihalo- and dihalo-4-methyl-5-hydroxy-2(5H)-furanones, respectively, notwithstanding substitutions by bromine or chlorine. Trihalides lacking the C-5 hydroxyl group possessed mutagenicities of the order of 10(2) rev/nmol, while hydroxyl group absence in the dihalides resulted in potency levels of slightly less than 10 rev/ nmol. Pairwise comparisons of compound mutagenicities showed that overall the C-5 H-by-OH replacement and, next in importance, increasing the number of C-6 halogens from one to two resulted in the greatest enhancements of mutagenicities. However, in comparing compound pairs within two different sets of four di- and trihalides, it was observed that replacement of a C-5 H by OH enhanced mutagenicity more for the dihalides than the trihalides indicating that increasing the C-6 halogen number simultaneously with replacing C-5 H by OH results in a nonlinear, additive enhancement. For fewer than half of the compound pairs compared, changing the C-6 halogen from chlorine to bromine resulted in small increases in mutagenicity, and for the remaining compound pairs, no increase could be discerned. This result points to the relative unimportance of only C-6 halogen type as a determinant of mutagenicity. Similarly, no impact on mutagenicity was observed for changing only the halogen type attached to C-3.

Presence and regional variation in peptide-containing nerves in the human ureter.

The occurrence, distribution and regional variation of neurones immunoreactive for the neuropeptides, vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), enkephalin (ENK), calcitonin gene-related peptide (CGRP), and substance P (SP) were investigated in human ureters by indirect immunohistochemistry. In addition, immunoreactivities to tyrosine hydroxylase (TH), a marker of noradrenergic neurones and to protein gene product (PGP) 9.5, a general marker of neurones, were also studied. Neurones displaying PGP-, NPY-, VIP- and TH-like immunoreactivity (-LIR) provided a rich innervation to the smooth muscle and blood vessels of the ureter, where they formed dense muscular and perivascular nerve plexuses. In contrast, there was only a moderate to sparse innervation by SP and CGRP-LIR neurones, most of which were distributed to blood vessels and to the sub mucosal layer, and only rarely to smooth muscle bundles. No ENK-LIR was detected in this study. Nerve fibre bundle densities were estimated for each of the localized neurochemicals according to a method described. NPY-LIR nerve fibre bundles were found to account for 80% of the total nerve fibre bundles (i.e. PGP-LIR) in the ureter. On the other hand, TH-LIR and VIP-LIR nerve fibre bundles each accounted for 50% of the total ureteral innervation, whereas SP- and CGRP-LIR nerve fibre bundles each comprised 20% of the total innervation. The abundance and pattern of tissues innervated by these immunoreactive neurones is consistent with the view that some of these neuropeptide substances co-exist with other peptide substances and/or with other known neurotransmitters, such as noradrenaline or acetylcholine. A gradient of innervation was found to exist for all the neurochemicals demonstrated in the ureter, whereby the lower ureter receives a greater density of innervation than the upper ureter. This finding suggests the human ureter is primarily innervated by fibres arising from or via the lower pelvis, i.e. the pelvic plexus. It also supports the view that the lower ureter may perform an important physiological role, such as coordinating the tone of this region during bladder filling and emptying.

Hemicellulose bioconversion to polyanionic heteropolysaccharides.

Anionic polysaccharides, traditionally obtained from plant or algal sources, have a variety of commercial uses. Such gums from microorganisms have received increased recent interest. We have initiated a program to investigate the bioconversion of pentosans to rheologically useful anionic extracellular polysaccharides (AEPS). A number of earlier-described species, including Cryptococcus laurentii, Klebsiella pneumoniae, Arthrobacter viscosus, and Pseudomonas ATCC 31260, appear to have potential in this regard. These organisms can individually convert either xylose, enzymatic oligomeric hemicellulose digests, dilute mineral acid hemicellulose ("TVA") hydrolysates, or a five-monosaccharide mixture simulating sulfite process liquors to AEPS. The formation parameters, compositions, mol-wt distributions, and the intrinsic viscosities of these purified AEPS are exemplified. Substitution of pentose as the major substrate for glucose can result in changes in mol-wt distribution or in the percentage of noncarbohydrate substituents in some AEPS. Pursuit of these observations may lead to interesting structure-property relationships and toward rheological applications for pentosan-derived AEPS.