Comparative electron microscopic visualization of the lung alveolar epithelial glycocalyx with different staining and labeling methods.

The alveolar surface of the lung is lined by an epithelium consisting of type I (AECI) and type II alveolar epithelial cells (AECII). This epithelium is covered by a liquid alveolar lining layer (ALL). Besides intra-alveolar surfactant, ALL also contains the alveolar epithelial glycocalyx on the apical side of AECI and AECII. To better understand the alveolar epithelial glycocalyx, its ultrastructural visualization by transmission electron microscopy is required. The aim of this study was to systematically re-evaluate routine cytochemical methods for visualization of the alveolar epithelial glycocalyx and specifically its glycan components. For this purpose, we used chemical fixation by vascular perfusion with aldehydes as a common routine approach in mice. After fixation, staining is needed for glycocalyx visualization. Cytochemical staining agents such as alcian blue, ruthenium red, and lanthanum nitrate were compared. In addition, SNL (Sambucus nigra lectin) and UEA1 (Ulex europaeus agglutinin I) were used for sialic acid and fucose-specific labeling. Alcian blue showed the strongest staining, with cloud-like structures, whereas ruthenium red appeared as thread-like structures. On the other hand, lanthanum nitrate did not stain the alveolar epithelial glycocalyx. For specific sialic acid and fucose labeling, both lectins presented a specific signal. In conclusion, these methods can be used routinely for assessing ultrastructural changes of the alveolar epithelial glycocalyx in experimental in vivo models under different physiological and pathological conditions. In addition, cytochemical staining by tissue massage and post-embedding lectin labeling after vascular perfusion support 3R (reduction, refinement, replacement) principles of animal welfare.

Age-related decline in goblet cell numbers and mucin content of the human colon: Implications for lower bowel functions in the elderly.

BACKGROUND & AIMS: Older people experience a greater incidence of lower bowel disorders, including constipation. Causes can include factors associated with growing older, such as use of medications or disease, but compounded by degenerative changes within the bowel wall. It has been suggested that the latter is exacerbated by loss of an effective mucosal barrier to luminal contents. In human colon, little is known about the impact of ageing on key components of this barrier, namely the goblet cells and mucin content. METHODS: Changes in the number of goblet cells and density of mucin content were investigated in macroscopically normal human ascending (AC; n = 13) and descending (DC; n = 14) colon from elderly (≥ 67 years) and younger adults (60 years and below). Samples were serially sectioned and stained for haematoxylin and eosin to assess tissue morphology, and alcian blue periodic acid Schiff (ABPAS) and MUC-2 antibody to identify goblet cells producing mucins. New procedures in visualization and identification of goblet cells and mucin contents were employed to ensure unbiased counting and densitometric analysis. RESULTS: Compared with the younger adults, the numbers of goblet cells per crypt were significantly lower in the elderly AC (72 ± 1.2 vs 51 ± 0.5) and DC (75 ± 2.6 vs. 54 ± 1.9), although this reduction did not reach statistical significance when assessed per mucosal area (AC: P = 0.068; DC: P = 0.096). In both regions from the elderly, numerous empty vesicles (normally containing mucins) were observed, and some areas of epithelium were devoid of goblet cells. Thus, the density of mucin content per unit mucosal area were significantly reduced with age. CONCLUSIONS: Ageing could result in a reduced number of goblet cells and development of degenerative changes in mucin production. Together, these have implications for the mucus barrier function in the colon of elderly individuals.

Differential Expression of Mucin in Salivary Gland Tumours.

Background and Objectives: Mucin has been implicated via various mechanisms in the development and growth of tumour cells. However, mucin expression studies in salivary gland tumours are limited, especially with samples from minor salivary glands. This study aims to investigate and compare mucin expression in benign and malignant salivary gland tumours of minor and major salivary gland origins. Materials and Methods: Special stains were used to stain neutral mucin (Periodic acid Schiff), sialomucin (Alcian Blue) and sulfomucin (Aldehyde Fuschin) within tissues from six normal salivary glands and 73 salivary gland tumours including 31 pleomorphic adenomas, 27 mucoepidermoid carcinomas, and 15 adenoid cystic carcinomas. A semi-quantitative approach was used to evaluate mucin expression within ductal lumens. Sialomucin was the most expressed mucin in all salivary gland tumours, regardless of origin. Results: A significant difference was observed in the mucin expression between benign and malignant salivary gland tumours, as pleomorphic adenoma showed three times significantly higher expression of sialomucin compared to mucoepidermoid carcinoma and adenoid cystic carcinoma (p = 0.028). Pleomorphic adenomas of major glands showed 42 times significantly higher expression of sialomucin compared to those of minor glands (p = 0.000). Conclusions: Sialomucin content in pleomorphic adenomas of major glands was vastly increased compared to that in minor glands. Differential sialomucin expression in benign and malignant salivary gland tumours suggests a role in diagnosing of borderline salivary gland tumours.

Empirical testing of cryoconite granulation: Role of cyanobacteria in the formation of key biogenic structure darkening glaciers in polar regions.

Cryoconite, the dark sediment on the surface of glaciers, often aggregates into oval or irregular granules serving as biogeochemical factories. They reduce a glacier's albedo, act as biodiversity hotspots by supporting aerobic and anaerobic microbial communities, constitute one of the organic matter (OM) sources on glaciers, and are a feeder for micrometazoans. Although cryoconite granules have multiple roles on glaciers, their formation is poorly understood. Cyanobacteria are ubiquitous and abundant engineers of cryoconite hole ecosystems. This study tested whether cyanobacteria may be responsible for cryoconite granulation as a sole biotic element. Incubation of Greenlandic, Svalbard, and Scandinavian cyanobacteria in different nutrient availabilities and substrata for growth (distilled water alone and water with quartz powder, furnaced cryoconite without OM, or powdered rocks from glacial catchment) revealed that cyanobacteria bind mineral particles into granules. The structures formed in the experiment resembled those commonly observed in natural cryoconite holes: they contained numerous cyanobacterial filaments protruding from aggregated mineral particles. Moreover, all examined strains were confirmed to produce extracellular polymeric substances (EPS), which suggests that cryoconite granulation is most likely due to EPS secretion by gliding cyanobacteria. In the presence of water as the only substrate for growth, cyanobacteria formed mostly carpet-like mats. Our data empirically prove that EPS-producing oscillatorialean cyanobacteria isolated from the diverse community of cryoconite microorganisms can form granules from mineral substrate and that the presence of the mineral substrate increases the probability of the formation of these important and complex biogeochemical microstructures on glaciers.

Optimization of Alcian blue pH 1.0 histo-staining protocols to match mass spectrometric quantification of sulfomucins and circumvent false positive results due to sialomucins.

Sulfomucins are in some body locations and species a normal occurrence, whereas in other situations, are a sign of pathology. Sulfomucin content on histological sections and isolated material is frequently analyzed with Alcian blue staining at pH 1.0. However, since the stain detects the charge, a high density of other charged molecules, such as sialic acids, has potential to impede specificity. Here, we compared the outcome from four staining protocols with the level of sulfation determined by liquid chromatography-tandem mass spectrometric analysis on samples from various tissues with variable sulfation and sialylation levels. We found that a protocol we designed, including rinsing with MetOH and 0.5 M NaCl buffer at pH 1.0, eliminates the false positive staining of tissues outperforming commonly recommended solutions. In tissues with low-to-moderately sulfated mucins (e.g. human stomach and salmonid epithelia), this method enables accurate relative quantification (e.g. sulfate scoring comparisons between healthy and diseased tissues), whereas the range of the method is not suitable for comparisons between tissues with high sulfomucin content (e.g. pig stomach and colon).

[Transthyretin amyloid cardiomyopathy. Features of histological diagnosis: study design].

AIM: To compare efficiency and specific features of transthyretin amyloid staining by different histological dyes and thus to assess their suitability for diagnostic purposes. MATERIALS AND METHODS: Samples of left and right heart ventricles were taken from patients over 70 years-old of both genders (n=10) with immunohistochemically verified transthyretin amyloidosis (ATTR). All samples were stained with Congo red, Alcian blue, toluidine blue and methylene violet. RESULTS: Specificity and sensitivity of Congo red staining was comparable to those of immunohistochemical staining. For verification of amyloid presence after Congo red staining one could use fluorescent microscopy instead of polarization microscopy. It allows a more accurate diagnosis of amyloidosis. Confocal microscopy with spectral unmixing improves detection sensitivity of amyloid by elimination of background fluorescence of muscle tissue and autofluorescence of lipofuscin. Alcian blue staining gives the same result as Congo red. In addition, its less labor-intensive and free of false-positive and false-negative results caused by final processing of slide preparation. Toluidine blue and methylene violet develop metachromatic staining upon binding to transthyretin fibrils, likely due to specific biochemical features of these fibrils. CONCLUSION: The most reliable method for histochemical diagnosis of ATTR is the Congo red staining with subsequent analysis using fluorescence or confocal microscopy. For diagnostic screening, the use of Sodium sulphate-Alcian blue staining method is highly promising. Metachromatic stains are less effective for ATTR diagnosis.

Three-dimensional electron microscopy for endothelial glycocalyx observation using Alcian blue with silver enhancement.

Glycocalyx (GCX) is a thin layer of negatively charged glycoproteins that covers the vascular endothelial surface and regulates various biological processes. Because of the delicate and fragile properties of this structure, it is difficult to detect GCX morphologically. We established a simple method for a three-dimensional visualization of endothelial GCX using low-vacuum scanning electron microscopy (LVSEM) on formalin-fixed paraffin-embedded (FFPE) sections. Mouse kidney tissue was fixed with 10% buffered formalin containing 1% Alcian blue (ALB) via perfusion and immersion. FFPE sections were observed by light microscopy (LM) and LVSEM, and formalin-fixed epoxy resin-embedded ultrathin sections were observed by transmission electron microscopy (TEM). The endothelial GCX from various levels of kidney blood vessels was stained blue in LM and confirmed as a thin osmiophilic layer in TEM. In LVSEM, the sections stained by periodic acid methenamine silver (PAM) revealed the endothelial GCX as a layer of dense silver-enhanced particles, in both the samples fixed via perfusion and immersion. Correlative light and electron microscopy (CLEM) revealed the fine visible structure of endothelial GCX. This simple method using FFPE samples with ALB will enable the three-dimensional evaluation of endothelial GCX alterations in various human diseases associated with endothelial injury in future studies.

A Histopathology-based Assessment of Biological Behavior in Oral Hyalinizing Odontogenic Tumors and Bone Lesions by Differential Stains.

AIM: Odontogenic tumors (OTs) and bone lesions of the oral cavity present diverse histological features and varying clinical behavior that makes predicting their biologic behavior difficult. The research undertaken in the current study aims to predict the biological behavior of oral hyalinizing odontogenic and bone lesions (OHO-BL) for the first time by employing four differential stains with clinicopathologic correlation. MATERIALS AND METHODS: The study was performed on retrospectively diagnosed formalin-fixed paraffin-embedded cases of OTs (n = 53) and bone lesions (n = 10). The severity of hyalinization (SOH) was assessed from stained tissue sections. Polarizing microscopy was used to analyze hyalinization in tissues stained with differential special stains, namely periodic acid-Schiff (PAS), Safranin-O, Alcian Blue, and Picrosirius red. SOH was also analyzed for possible correlation with recurrence and clinicopathologic correlation in OHO-BL. RESULTS: Intense staining was observed with PAS, Alcian Blue, and Safranin-O in OTs with increased SOH with a statistical significance. Polarizing greenish yellow color correlated significantly with the recurrence potential of the OT group. Recurrence in individual lesions of the OT group showed a statistically significant association with SOH. Such individual correlation was not observed in bone diseases. CONCLUSION: PAS, Alcian Blue, Safranin-O, and Picrosirius red are reliable stains to assess hyalinization in OHO-BL. Picrosirius red-polarizing microscopy is a dependable tool for identifying recurrent odontogenic lesions. CLINICAL SIGNIFICANCE: SOH can be considered a histological predictor of aggressive biologic behavior in oral hyalinizing odontogenic lesions that can enable the surgeon to arrive at an appropriate management protocol.

A Histopathology-based Assessment of Biological Behavior in Oral Hyalinizing Extraosseous Lesions by Differential Stains.

AIM: The assessment of hyalinization to determine aggressive behavior in oral pathological lesions is a scarcely researched field that requires further exploration. The current study aims to predict the biological behavior of oral hyalinizing extraosseous lesions (OHEOL) by employing four differential stains with clinicopathologic correlation. MATERIALS AND METHODS: The study was performed on retrospectively diagnosed formalin-fixed paraffin-embedded cases of salivary gland tumors (SGTs) (n = 13), benign soft tissue (BST) lesions (n = 24), and oral submucous fibrosis (OSMF) (n = 53). The hematoxylin and eosin-stained sections were analyzed for the severity of hyalinization (SOH). Differential stains periodic acid Schiff (PAS), Alcian blue, safranin O, and picrosirius red with polarizing microscopy were used to assess the components of hyalinized tissue. The SOH was correlated with differential staining characteristics and clinicopathologic features to analyze possible correlation with aggressive potential in BST, advancement of disease in OSMF, and recurrence in SGT. RESULTS: Intensity of picrosirius red stain significantly correlated with SOH of SGTs (p = 0.044). The intensity of PAS stain (p = 0.040), picrosirius red polarizing greenish-yellow color (p = 0.002), and pattern of distribution of picrosirius red (p = 0.023) significantly correlated with recurrence of SGTs. The intensity of differential stains increased with the SOH in BST lesions indicating their correlation with SOH. The intensity (p = 0.008) and pattern (p = 0.010) of Alcian blue staining and intensity of safranin O stain (p = 0.003) significantly correlated with SOH in OSMF. Picrosirius red polarizing color reddish and yellowish red (p = 0.002) significantly correlated with SOH distinguishing early and advanced OSMF. CONCLUSION: Picrosirius red and PAS stains are reliable indicators of SOH and recurrence potential in SGT. Alcian blue, safranin O, and picrosirius red polarizing colors enable detection of SOH and accurately distinguish early from advanced OSMF. CLINICAL SIGNIFICANCE: SOH can be considered as a histological predictor of aggressive biologic behavior in OHEOL. These findings will result in appropriate management protocols.

Evaluation of Better Staining Method among Hematoxylin and Eosin, Giemsa and Periodic Acid Schiff-Alcian Blue for the Detection of Helicobacter pylori in Gastric Biopsies.

BACKGROUND: This study was undertaken to evaluate the preferred method (Giemsa or periodic acid Schiff-Alcian blue [PAS-AB] stains) of detecting Helicobacter pylori (H. pylori) in gastric mucosal biopsies in terms of sensitivity, specificity and applicability. To the best of my knowledge, this is the first report comparing Giemsa and PAS-AB staining for the detection of H. pylori in such biopsies. METHODS: The formalin-fixed paraffin-embedded blocks of 49 gastric biopsies from different patients were collected from the archive of anatomical pathology at King Abdulaziz Medical City, National Guard, Riyadh, Saudi Arabia. From each block, three slides were prepared and analysed using the hematoxylin and eosin (H&E), Giemsa and PAS-AB stains to detect the presence/absence of H. pylori, and the results were compared in terms of sensitivity, specificity and applicability. RESULTS: The majority of the biopsies in this study showed antrum-type gastric mucosa. Only 15 biopsies showed active gastritis, whereas the rest showed chronic gastritis. Three biopsies showed intestinal metaplasia. All were detected by PAS-AB stain, but only two-thirds were detected by H&E stain. Fifteen gastric biopsies showed H. pylori infection in general and in 13 of them, active gastritis cases were discovered. Fourteen out of these 15 H. pylori infection cases were detected by Giemsa stain, whereas only 13 cases were detected by H&E stain. PAS-AB stain showed the worst results since it demonstrated only 40% sensitivity and 67.65% specificity in H. pylori detection. CONCLUSION: Giemsa stain has better sensitivity and specificity in gastric H. pylori infection detection than PAS-AB. Therefore, using PAS-AB stain to detect H. pylori infection is not recommended.

The Histochemical Alterations of Mucin in Colorectal Carcinoma Quantified by Two Efficient Algorithms of Digital Image Analysis.

The practical use of knowledge on the diagnostic-prognostic role of polysaccharide components of mucins in colorectal cancer (CRC) has been difficult, due to the number of histochemical (HC) reaction types, as well as lack of standard methods of computer-assisted analysis of tissue expression of these molecules. Using two algorithms of digital image analysis (by application of Image-Pro Premier and our originally designed program Filter HSV), we evaluated the expression of polysaccharides in tissue samples of CRC patients (n = 33), and fragments of normal colorectal tissue from the same patients (control) using periodic acid Schiff reaction (PAS) (neutral mucins) and alcian blue staining (AB) (acidic mucins). Our results indicate lower expression of the PAS+ and AB+ mucins in CRC, as compared to the control samples. The higher expression of PAS+ polysaccharides was detected in flat tumors than in protruded CRC, while higher AB+ mucins expression was a feature of mucinous CRC subtypes. Positive correlation between mutual PAS+ and AB+ expression, as well as correlations with glucose concentration (PAS+ mucins), and hemoglobin level (AB+ mucins) were observed exclusively in unchanged colorectal samples (control). Both algorithms of digital image analysis (smart segmentation and Filter HSV) work properly and can be used interchangeably in daily practice of pathologists, as useful tools of quantitative evaluation of HC reaction in both normal and cancerous tissues.

Expression analysis of limb element markers during mouse embryonic development.

BACKGROUND: While data regarding expression of limb element and tissue markers during normal mouse limb development exist, few studies show expression patterns in upper and lower limbs throughout key limb development stages. A comparison to normal developmental events is essential when analyzing development of the limb in mutant mice models. RESULTS: Expression patterns of the joint marker Gdf5, tendon and ligament marker Scleraxis, early muscle marker MyoD1, and blood vessel marker Cadherin5 (Cdh5) are presented during the most active phases of embryonic mouse limb patterning. Anti-neurofilament staining of developing nerves in the fore- and hindlimbs and cartilage formation and progression also are described. CONCLUSIONS: This study demonstrates and describes a range of key morphological markers and methods that together can be used to assess normal and abnormal limb development. Developmental Dynamics 247:1217-1226, 2018. © 2018 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Variations in internal structure, composition and protein distribution between intra- and extra-articular knee ligaments and tendons.

Tendons and ligaments play key roles in the musculoskeletal system in both man and animals. Both tissues can undergo traumatic injury, age-related degeneration and chronic disease, causing discomfort, pain and increased susceptibility to wider degenerative joint disease. To date, tendon and ligament ultrastructural biology is relatively under-studied in healthy, non-diseased tissues. This information is essential to understand the pathology of these tissues with regard to function-related injury and to assist with the future development of tissue-engineered tendon and ligament structures. This study investigated the morphological, compositional and extracellular matrix protein distribution differences between tendons and ligaments around the non-diseased canine stifle joint. The morphological, structural characteristics of different regions of the periarticular tendons and ligaments (the intra-articular anterior cruciate ligament, the extra-articular medial collateral ligament, the positional long digital extensor tendon and energy-storing superficial digital flexor tendons) were identified using a novel semi-objective histological scoring analysis and by determining their biochemical composition. Protein distribution of extracellular matrix collagens, proteoglycans and elastic fibre proteins in anterior cruciate ligament and long digital extensor tendon were also determined using immunostaining techniques. The anterior cruciate ligament was found to have significant morphological differences in comparison with the other three tissues, including less compact collagen architecture, differences in cell nuclei phenotype and increased glycosaminoglycan and elastin content. Intra- and interobserver differences of histology scoring resulted in an average score 0.7, indicative of good agreement between observers. Statistically significant differences were also found in the extracellular matrix composition in terms of glycosaminoglycan and elastin content, being more prominent in the anterior cruciate ligament than in the other three tissues. A different distribution of several extracellular matrix proteins was also found between long digital extensor tendon and anterior cruciate ligament, with a significantly increased immunostaining of aggrecan and versican in the anterior cruciate ligament. These findings directly relate to the different functions of tendon and ligament and indicate that the intra-articular anterior cruciate ligament is subjected to more compressive forces, reflecting an adaptive response to normal or increased loads and resulting in different extracellular matrix composition and arrangement to protect the tissue from damage.

Glycosaminoglycans detection methods: Applications of mass spectrometry.

Glycosaminoglycans (GAGs) are long blocks of negatively charged polysaccharides. They are one of the major components of the extracellular matrix and play multiple roles in different tissues and organs. The accumulation of undegraded GAGs causes mucopolysaccharidoses (MPS). GAGs are associated with other pathological conditions such as osteoarthritis, inflammation, diabetes mellitus, spinal cord injury, and cancer. The need for further understanding of GAG functions and mechanisms of action boosted the development of qualitative and quantitative (alcian blue, toluidine blue, paper and thin layer chromatography, gas chromatography, high pressure liquid chromatography, capillary electrophoresis, 1,9-dimethylmethylene blue, enzyme linked-immunosorbent assay, mass spectrometry) techniques. The availability of quantitative techniques has facilitated translational research on GAGs into the medical field for: 1) diagnosis, monitoring, and screening for MPS; 2) analysis of GAG synthetic and degradation pathways; and 3) determination of physiological and pathological roles of GAGs. This review provides a history of development of GAG assays and insights about the use of tandem mass spectrometry and its applications for GAG analysis.

Temporal Change of Alcian Blue-Stained Primo Vascular System in Lymph Vessels of Rats.

This study aims to investigate the temporal change of a vascular system now known as the primo vascular system (PVS). We used Alcian blue (AB) dye for imaging the distribution of the PVS in lymphatic vessels. The target lymph vessels were chosen as they are easily accessible from the skin, and long-term observation is possible with intact physiological conditions due to a minimal surgical procedure. AB solution was injected into the inguinal lymph node and the target lymph vessels were located along the superficial epigastric vessels. The imaging system allowed processing for extraction of images showing changes in the AB intensity of the visualized PVS components. This newly developed procedure can be used for further study on various dynamic processes of PVS in lymph vessels.

Form and function of the intervertebral disc in health and disease: a morphological and stain comparison study.

Multiple histologic measurements are commonly used to assess degenerative changes in intervertebral disc (IVD) structure; however, there is no consensus on which stains offer the clearest visualization of specific areas within the IVD. The objective of this study was to compare multiple tinctorial stains, evaluate their ability to highlight structural features within the IVD, and investigate how they influence the capacity to implement a degeneration scoring system. Lumbar IVDs from seven human autopsy specimens were stained using six commonly used stains (Hematoxylin/Eosin, Toluidine Blue, Safranin-O/Fast Green, Extended FAST, modified Gomori's Trichrome, and Picrosirius Red Alcian Blue). All IVDs were evaluated by three separate graders to independently determine which stains (i) were most effective at discerning different structural features within different regions of the IVDs and (ii) allowed for the most reproducible assessment of degeneration grade, as assessed via the Rutges histological scoring system (Rutges et al. A validated new histological classification for intervertebral disc degeneration. Osteoarthritis Cartilage, 21, 2039-47). Although Trichrome, XFAST and PR/AB stains were all effective at highlighting different regions of whole IVDs, we recommend the use of PR/AB because it had the highest degree of rater agreement on assigned degeneration grade, allowed greater resolution of degeneration grade, has an inferential relationship between color and composition, and allowed clear differentiation of the different regions and structural disruptions within the IVD. The use of a standard set of stains together with a histological grading scheme can aid in the characterization of structural changes in different regions of the IVD and may simplify comparisons across the field. This collection of human IVD histological images highlights how IVD degeneration is not a single disease but a composite of multiple processes such as aging, injury, repair, and disease, each of which are unique to the individual.

Association of heterotrophic bacteria with aggregated Arthrospira platensis exopolysaccharides: implications in the induction of axenic cultures.

Inducing an axenic culture of the edible cyanobacterium Arthrospira (Spirulina) platensis using differential filtration alone is never successful; thus, it has been thought that, in non-axenic cultures, a portion of contaminating bacteria is strongly associated with Arthrospira cells. However, examination of the behavior of these bacteria during filtration revealed that they were not associated with Arthrospira cells but with aggregates of exopolysaccharides present in the medium away from the Arthrospira cells. Based on this finding, a rapid and reliable method for preparing axenic trichomes of A. platensis was established. After verifying the axenicity of the resulting trichomes on enriched agar plates, they were individually transferred to fresh sterile medium using a handmade tool, a microtrowel, to produce axenic cultures. With this technique, axenic cultures of various A. platensis strains were successfully produced. The technique described in this study is potentially applicable to a wider range of filamentous cyanobacteria.

Amniotic components in the uterine vasculature and their role in amniotic fluid embolism.

AIM: To evaluate whether the presence of amniotic components in the maternal uterine vasculature could be a specific pathological indicator for amniotic fluid embolism (AFE). METHODS: Medical records of patients treated between January 2006 and March 2013 were retrospectively examined to identify patients who underwent post-partum hysterectomy or autopsy due to maternal death. Three subjects with AFE with disseminated intravascular coagulation (DIC)-type post-partum hemorrhage (PPH), and 13 non-AFE subjects were included in analysis. Histochemical staining using hematoxylin-eosin (HE) and alcian blue, and immunohistochemical staining for sialyl-Tn were conducted to detect amniotic components in the maternal uterine vasculature. RESULTS: Alcian blue was positive for amniotic components in the uterine vasculature of all subjects with AFE and of several subjects without AFE. Similarly, HE and sialyl-Tn were negative in some AFE subjects and positive in some non-AFE subjects. CONCLUSIONS: The presence of maternal intravascular fetal material is not a specific indicator for AFE with DIC-type PPH. Therefore, the presence of fetal components in the uterine vasculature is unlikely to be a definitive indicator for AFE.

Endothelial glycocalyx layer in the aqueous outflow pathway of bovine and human eyes.

The glycocalyx layer on the vascular endothelium is known to have an important role as a transport barrier and in the mechanotransduction of fluid shear stress. The detailed structure and distribution of the glycocalyx in the bovine and human aqueous humor outflow pathways has not yet been reported. The purpose of this study was to determine whether this layer exists in the bovine and human aqueous outflow pathways and to compare the distribution and thickness therein. Enucleated bovine (N = 4) and human (N = 4) eyes were fixed using Alcian Blue to preserve the glycocalyx. The glycocalyx distribution and thickness (in regions where it was seen) were measured on the trabecular beams (TM), Schlemm's canal (SC)/aqueous plexus (AP), and collector channels (CC). The glycocalyx, which appears as a layer of hair-like brushes, coats the surface of the endothelium non-uniformly in the bovine and human aqueous outflow pathways with a thickness in bovine eyes of 68-122 nm and in human eyes of 52-166 nm (25th to 75th percentiles). The distribution of the glycocalyx in different regions of the outflow pathway is not the same between bovine and human eyes. In both species, the glycocalyx was most uniform in the CCs. Less coverage of glycocalyx was found in the AP than the TM in bovine eyes, while more coverage was found in SC than the TM in human eyes. Most interestingly, glycocalyx was also found filling most pores of the endothelium of AP/SC in both bovine and human eyes. Glycocalyx was usually not found coating the inner membranes of the giant vacuoles (GVs); however, in GVs with a visible pore, glycocalyx was frequently observed on the inner membranes of the GVs. Based on our findings and those from the vascular endothelium, it is likely that the glycocalyx in SC plays a role in transduction of shear stress and perhaps regulation of outflow resistance.

Quantitation of Mucin by Densitometry of an Alcian Blue-Stained Membrane.

It is a challenging task to quantify mucin using conventional protein quantification methods due to the large number of glycans attached to the peptide, which make up approximately 50-90% of its molecular weight. To address this issue, we propose a simple quantification method that involves spotting mucins onto a membrane and staining them with Alcian blue.