Standard Staining Research Articles

Opacification of Hydrophilic Acrylic Intraocular Lenses: Overview of Laboratory Methods for Histological Analysis and Replication of IOL Calcification.

Opacification of intraocular lenses (IOLs) due to material changes is a serious complication that can compromise the good visual outcomes of uncomplicated cataract surgery. In hydrophobic acrylic IOLs, opacification can result from glistening formation, while in hydrophilic acrylic IOLs, there is a risk of calcification due to the formation of calcium phosphates within the polymer. Over time, various methods have been developed to investigate calcification in hydrophilic acrylic IOLs. The aim of this article is to provide an overview of standard histological staining and models used to simulate IOL calcification. Histological staining can be used to detect calcification and assess the extent of crystal formation. The development of in vivo and in vitro replication models has helped to identify the underlying pathomechanisms of calcification. In vivo models are suitable for assessing the biocompatibility of IOL materials. Bioreactors as an in vitro model can be used to investigate the kinetics of crystal formation within the polymer. The replication of IOL calcification under standardized conditions using electrophoresis allows for the comparison of different lens materials with respect to the risk of calcification. The combination of different analytical and replication methods can be used in the future to further investigate the pathomechanisms of calcium phosphate crystal formation and the influence of risk factors. This may help to prevent calcification of hydrophilic acrylic IOLs and associated explantation and complications.

Chromosome Diversity and Evolution of the Endemic Malagasy Velvet Geckos of the Genus Blaesodactylus (Reptilia, Gekkonidae).

We performed a molecular and phylogenetic analysis and a comparative cytogenetic study with standard karyotyping, silver staining (Ag-NOR) and sequential C-banding + Giemsa, + fluorochromes on several Blaesodactylus samples. The phylogenetic inference retrieved two main clades, the first comprises B. victori, B. microtuberculatus and B. boivini, while the second includes B. sakalava, B. antongilensis and B. ambonihazo. The available samples of B. sakalava form two different clades (here named B. sakalava clade A and clade B), which probably deserve a taxonomic re-evaluation. We found a karyological variability in Blaesodactylus in terms of chromosome number (2n = 40-42), morphology, location of NORs, and heterochromatin distribution pattern. Blaesodactylus antongilensis and B. sakalava clade A and B showed a karyotype of 2n = 40 mostly telocentric chromosomes. Pairs 1 and 6 were metacentric in B. sakalava clade A and B, while pair 1 was composed of subtelocentric/submetacentric elements in B. antongilensis. In contrast, B. boivini displayed a karyotype with 2n = 42 only telocentric chromosomes. NORs were on the first chromosome pair in B. boivini, and on the second pair in B. antongilensis. Adding our data to those available from the literature on evolutionarily related species, we highlight that the chromosome diversification in the genus probably proceeded towards a progressive reduction in the chromosome number and the formation of metacentric elements.

Smartphone‐based rapid and visual pathological diagnosis of glioma using perovskite probes

AbstractHistopathology plays a great role in diagnosing various diseases, which is considered as a golden standard for tumor identification. The tissue constituents must be stained by visible labels for microscopic analysis by medical experts. However, this process is time‐consuming, labor‐intensive, and expensive, which requires rapid pathological approaches for diagnosis in the operating room. Here, we present an easy‐to‐process and high‐performance perovskite biological probes for rapid and visual pathological diagnosis of glioma. Perovskite quantum dots can be encapsulated by the copolymer into nanocrystals (PNCs) with a diameter of 100 nm, which is modified with chlorotoxin to achieve the specific recognition of glioma. Benefiting from the super photoluminescence quantum yield (above 93%) of EVA@PNCs aqueous solution, the glioma can be clearly imaged and captured via a smartphone under the excitation of a handheld UV lamp. To demonstrate the visualization and efficiency of PNC probes, different malignant grades of brain tumor sections can be distinguished in no more than 5 min. This strategy provides a general auxiliary diagnosis platform for achieving the histopathology analysis near the operating bed, which is currently not feasible with standard histochemical staining methods.image

THE EFFECT OF ORALLY ADMINISTERED COENZYME Q10 ON THE VIABILITY OF RANDOM SKIN FLAP IN NICOTINE EXPOSED WISTAR RATS

Introduction : Nicotine was assumed to be the cause of compromised flap. The administration of antioxidants can eliminate such effect, and one of the most promising antioxidants is Coenzyme Q10. The research aims to find the effect of orally administered Coenzyme Q10 on the viability of random skin flaps in nicotine-exposed wistar rats. Method : Twenty-seven white rats (Rattus norvegicus) were assigned in three groups. The first group received no nicotine, while the other two groups were nebulized with nicotine for 4 consecutive weeks, and treated as random skin flaps. In third group, Coenzyme Q10 was administered for 7 days. Then, a standard histopathological staining and vascular endothelial growth factor (VEGF) measurement were performed on flap sample to examine the extent of neovascularization and VEGF cell expression. Result : The average number of capillaries in group I was 5.33 ± 1.323, in group II was 5.89 ± 0.782, and in group III was 7.78 ± 2.587. There was no significant difference (p = 0.317, 95% CI) in groups I and II of VEGF expression. However, significant differences were found in the intensity of VEGF (p = 0.009, 95% CI) in groups I and group III, and in groups II and group III (p = 0.011, 95% CI). Thus, the intensity was stronger on the subject with coenzyme Q10 compared with the other two groups. Conclusion: Coenzyme Q10 increases the viability of random flaps by increasing the number of capillaries and VEGF expression.

Multiplex immunofluorescence staining of coverslip-mounted paraffin-embedded tissue sections.

Animal and human tissues are used extensively in physiological and pathophysiological research. Due to both ethical considerations and low availability, it is essential to maximize the use of these tissues. Therefore, the aim was to develop a new method allowing for multiplex immunofluorescence (IF) staining of kidney sections in order to reuse the same tissue section multiple times. The paraffin-embedded kidney sections were placed onto coated coverslips and multiplex IF staining was performed. Five rounds of staining were performed where each round consisted of indirect antibody labelling, imaging on a widefield epifluorescence microscope, removal of the antibodies using a stripping buffer, and then re-staining. In the final round, the tissue was stained with hematoxylin/eosin. Using this method, tubular segments in the nephron, blood vessels, and interstitial cells were labeled. Furthermore, by placing the tissue on coverslips, confocal-like resolution was obtained using a conventional widefield epifluorescence microscope and a 60x oil objective. Thus, using standard reagents and equipment, paraffin-embedded tissue was used for multiplex IF staining with increased Z-resolution. In summary, this method offers time-saving multiplex IF staining and allows for the retrieval of both quantitative and spatial expressional information of multiple proteins and subsequently for an assessment of the tissue morphology. Due to the simplicity and integrated effectivity of this multiplex IF protocol, it holds the potential to supplement standard IF staining protocols and maximize use of tissue.

74 Secondary Immunohistochemical Analysis of Viable Cells as Determined by Hematoxylin and Eosin Staining

Abstract Introduction Accurate clinical differentiation of deep partial-thickness (DPT) and full-thickness (FT) wounds is essential in justifying appropriate treatments for heterogeneous burns. Only recently has non-invasive burn depth imaging been ground-truthed with tissue punch biopsies; an endeavor that will no doubt improve AI imaging algorithms in the near future. Prior literature supports unique apoptotic and necrotic properties of DPT and FT wounds, suggesting a use for not only standard stains (H&E), but apoptotic marker staining in assisting clinical diagnosis of burn depth, progression and/or conversion. Methods The aim of this study was to evaluate the presence of apoptotic cells in DPT and FT samples previously diagnosed by a burn biopsy algorithm (BBA-V2) for burn depth and severity by expert review of H&E-stained slides. Eight paired biopsies were collected from DPT and FT wounds in four patients within 72 hours of thermal injury and were immunofluorescently labeled for apoptosis markers by detection of deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and cleaved caspase-3 (CC-3) staining. Fluorescence intensity in dermal appendages within images was used to quantify signal at 40x magnification for CC-3. % positive TUNEL cells were graded by an investigator blinded to the burn severity. A higher grade indicated higher % positivity. TUNEL grading was evaluated by Chi square test. CC-3 intensity was evaluated by Student’s t-test. Results Across all four patients, FT burns had higher levels of CC-3 in dermal appendages compared to DPT burns (Cy3:DAPI ratios: 1.53 ± 0.28 vs. 0.75 ± 0.13; p=0.1074). Within each patient, all FT biopsy sites had significantly higher levels of CC-3 versus DPT biopsy sites (p< 0.05)(Table). DPT burns had a greater proportion of patients with increasing grades of TUNEL positive cells (DPT: Grade 1: 16%, Grade 2: 36%, Grade 3: 48%; FT: Grade 1: 42.3%, Grade 2: 23.1%, Grade 3: 34.6%, p=0.1178). Conclusions CC-3 and TUNEL levels within dermal appendages align with burn depth classification by H&E staining, wherein appendages graded as necrotic had higher levels of CC-3 and lower levels of TUNEL. Therefore, CC-3 and TUNEL are potential secondary histological markers for burn depth. Further analysis of isolated, individual dermal appendages is underway to identify what additional information this secondary marker may add to the diagnosis of burn depth. Applicability of Research to Practice For the first time there is access to a large tissue repository of biopsies from burn wounds. Although staining from these samples has been used to build diagnosis algorithms with success, much more can be learned from the cellular pathophysiology when secondary analyses are performed. These assays may help us understand the processes of burn conversion.

Diagnosis of Oral Cancers by Targeting VPAC Receptors: Preliminary Report.

Oral cancer is a major health problem. The study of exfoliative cytology material helps in the differentiation of premalignant and malignant alterations of oral lesions. The objective of this study was to assess the feasibility of detecting oral cancer by targeting genomic VPAC (combined vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide) receptors expressed on malignant oral cancer cells. All patients with suspected oral cavity cancers/lesions formed the study group. The samples from the oral cavity lesion or suspicious area were collected with a cytology brush. The harvested material was examined for malignant cells by 1. the standard PAP stain and 2. targeting the VPAC receptors on the cell surface using a fluorescent microscope. Similarly, malignant cells were identified from cells shed in oral gargles. A total of 60 patients with oral lesions were included in the study. The histopathological diagnosis was squamous cell carcinoma in 30 of these. The VPAC receptor positivity both on the brush cytology staining as well oral gargle staining was more sensitive than the brush cytology PAP staining. The accuracy of the various techniques was as follows, brush cytology PAP staining at 86.67%, brush cytology VPAC staining at 91.67% and oral gargle VPAC staining at 95%. This preliminary study validates our belief that malignant cells in the saliva can be identified by targeting the VPAC receptors. The test is simple, easy, non-invasive and reliable in the detection of oral cancers.

Modified Periodic Acid-Schiff (PAS) Is an Alternative to Safranin O for Discriminating Bone-Cartilage Interfaces.

Cartilage histomorphometry is often performed on decalcified, paraffin-embedded bone sections, which provide versatility in staining applications from basic morphology to immunohistochemistry. Safranin O is a cationic dye that binds to proteoglycans in cartilage and is routinely used to assess growth plate dynamics and/or fracture repair at bone-cartilage interfaces. When used with a counterstain such as fast green, safranin O can offer exquisite differentiation of cartilage from surrounding bone. However, various decalcification and processing methods can deplete proteoglycans, rendering inconsistent, weak, or absent safranin O staining with indiscriminate bone-cartilage boundaries. We sought to develop an alternative staining methodology that preserves the contrast of bone and cartilage in cases of proteoglycan depletion that can be applied when other cartilage stains are unsuccessful. Here, we describe and validate a modified periodic acid-Schiff (PAS) protocol that we developed using Weigert's iron hematoxylin and light green stains as an alternative to safranin O for discriminating bone-cartilage interfaces of skeletal tissues. This method provides a practical solution for differentiating bone and cartilage when safranin O staining is not detected after decalcification and paraffin processing. The modified PAS protocol can be useful for studies in which identification of the bone-cartilage interface is essential but may not be preserved with standard staining approaches. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Hematoxylin Nuclear Stain Reports Oxidative Stress via Near-Infrared Emission.

Hematoxylin & eosin (H&E) is the gold standard histological stain used for medical diagnosis and has been used for over a century. Herein, we examined the near-infrared II (NIR-II) fluorescence of this stain. We observed significant NIR-II emission from the hematoxylin component of the H&E stain. We found that the emission intensity, using the common aluminum(III) hematoxylin mordant, could be modulated by the availability of endogenous iron(III), and this emission intensity increased at higher oxidative stress. Our mechanistic investigations found that hematoxylin emission reported the nuclear translocation of the iron via the protein ferritin. In human tumor tissue samples, oxidative stress biomarkers correlated with hematoxylin NIR-II emission intensity. Emission response of the stain was also observed in human Alzheimer's disease brain tissue regions affected by disease progression, suggesting that ferritin nuclear translocation is preserved in these regions as an oxidative stress response. These findings indicate that NIR-II emission from the H&E stain provides a new source of redox information in tissues with implications for biomedical research and clinical practice.

Incidence and Evaluation of Foodborne Fungal Pathogens

Background: Fruits and vegetables provide an ideal condition for the growth of microorganisms. Cereals and bakery products serve as a valuable source of nutrients in the diet of many people and they provide an ideal condition for the growth of microorganisms too. Many fungal and bacterial species are responsible for the spoilage of fruits and vegetables. They contaminate fruits and vegetables and made them insufficient for consumption. Objective: In our present study, an investigation has been carried out to isolate and identify the fungal species which are responsible for the spoilage of some food items particularly bread, cheese, lemon and onion. Materials and method: A prospective study was conducted to know the incidence and to evaluate the common fungal pathogens are involved in the spoilage of different types of food products namely bakery product bread, milk product cheese, vegetables onion and lemon. The samples were collected from the spoiled bread, lemon, onion & cheese which were obtained from different shops in the local market. The samples were subjected to identification and isolation of fungal pathogens using the standard staining procedure and selective media. 5 fungal isolates were found to be most commonly occurring in all of the samples namely Aspergillus flavus, Aspergillus niger, Rhizopus, Mucor and Penicillium marneffei. Keywords: Aspergillus flavus, Aspergillus niger, Mucor and Penicillium marneffei.

A Histological and Clinical Study of MatriDerm® Use in Burn Reconstruction.

Dermal substitutes are well established in the reconstructive ladder. MatriDerm® (Dr. Otto Suwelack Skin & Health Care AG, Billerbeck, Germany) is a single-layer dermal substitute composed of a bovine collagen (type I, III, and V) and elastin hydrolysate, that allows for immediate split-thickness skin grafting (SSG). The aim of this study was to histologically characterize the integration of MatriDerm® when used during burns surgery reconstruction. Eight subjects with nine burn scars and one acute burn wound underwent reconstruction with MatriDerm® and an immediate SSG. MatriDerm® integration and skin graft take were assessed with serial biopsies performed at weeks 1, 2, 3, and 4 and months 2, 3, 6, 9, and 12. Biopsies were assessed with standard special stains and immunohistochemistry, and representative slides were imaged with a transmission electron microscope. Patient satisfaction and clinical scar outcome were assessed with the Vancouver Scar Scale and a patient questionnaire. Histological analysis showed similar stages of wound healing as shown in other dermal templates but on a different timescale. There is early evidence of vascularization and an inflammatory infiltrate in the first 2 weeks. MatriDerm® is resorbed earlier than other dermal substitutes, with evidence of resorption at week 3, to be completely replaced by a neodermis at 2 months. The use of MatriDerm® in reconstruction with immediate skin grafting is supported histologically with early evidence of vascularization to support an epidermal autograft. Future histological studies may help further characterize the ideal dermal substitute.

Determination of Intracellular Esterase Activity Using Ratiometric Raman Sensing and Spectral Phasor Analysis

Carboxylesterases (CEs) are a class of enzymes that catalyze the hydrolysis of esters in a variety of endogenous and exogenous molecules. CEs play an important role in drug metabolism, in the onset and progression of disease, and can be harnessed for prodrug activation strategies. As such, the regulation of CEs is an important clinical and pharmaceutical consideration. Here, we report the first ratiometric sensor for CE activity using Raman spectroscopy based on a bisarylbutadiyne scaffold. The sensor was shown to be highly sensitive and specific for CE detection and had low cellular cytotoxicity. In hepatocyte cells, the ratiometric detection of esterase activity was possible, and the result was validated by multimodal imaging with standard viability stains used for fluorescence microscopy within the same cell population. In addition, we show that the detection of localized ultraviolet damage in a mixed cell population was possible using stimulated Raman scattering microscopy coupled with spectral phasor analysis. This sensor demonstrates the practical advantages of low molecular weight sensors that are detected using ratiometric Raman imaging and will have applications in drug discovery and biomedical research.

Expanding the Chromosomal Evolution Understanding of Lygaeioid True Bugs (Lygaeoidea, Pentatomomorpha, Heteroptera) by Classical and Molecular Cytogenetic Analysis.

The Lygaeoidea comprise about 4660 species in 790 genera and 16 families. Using standard chromosome staining and FISH with 18S rDNA and telomeric (TTAGG)n probes, we studied male karyotypes and meiosis in 10 species of Lygaeoidea belonging to eight genera of the families Blissidae, Cymidae, Heterogastridae, Lygaeidae, and Rhyparochromidae. Chromosome numbers were shown to range from 12 to 28, with 2n = 14 being predominant. All species have an XY system and all but one have a pair of m-chromosomes. The exception is Spilostethus saxatilis (Lygaeidae: Lygaeinae); in another species of Lygaeinae, Thunbergia floridulus, m-chromosomes were present, which represents the first finding for this subfamily. All species have an inverted sequence of sex chromosome divisions ("post-reduction"). The 18S rDNA loci were observed on one or both sex chromosomes in Kleidocerys resedae and Th. floridulus, respectively (Lygaeidae), while on an autosomal bivalent in all other species. The rDNA loci tended to be close to the end of the chromosome. Using (TTAGG)n-FISH, we were able to show for the first time that the Lygaeoidea lack the canonical "insect" telomere motif (TTAGG)n. We speculate that this ancestral motif is absent from the entire infraorder Pentatomomorpha being replaced by some other telomere repeat motif sequences.

Defining the phenotypic spectrum of sporadic Creutzfeldt-Jakob disease MV2K: the kuru plaque type.

The current classification of sporadic Creutzfeldt-Jakob disease identifies six major subtypes mainly defined by the combination of the genotype at polymorphic codon 129 (methionine/M or valine/V) of the prion protein gene and the type (1 or 2) of misfolded prion protein accumulating in the brain (e.g. MM1, MM2, MV1, MV2, etc.). Here, we systematically characterized the clinical and histo-molecular features associated with the third prevalent subtype, the MV2 subtype with kuru plaques (MV2K), in the most extensive series collected to date. We evaluated neurological histories, cerebrospinal biomarkers, brain MRI and EEG results in 126 patients. The histo-molecular assessment included misfolded prion protein typing, standard histologic staining and immunohistochemistry for prion protein in several brain areas. We also investigated the prevalence and topographic extent of coexisting MV2-cortical features, the number of cerebellar kuru plaques and their effect on clinical phenotype. Systematic regional typing revealed a western blot profile of misfolded prion protein comprising a doublet of 19 and 20 kDa unglycosylated fragments, with the former more prominent in neocortices and the latter in the deep grey nuclei. The 20/19 kDa fragment ratio positively correlated with the number of cerebellar kuru plaques. The mean disease duration was exceedingly longer than in the typical MM1 subtype (18.0 versus 3.4 months). Disease duration correlated positively with the severity of pathologic change and the number of cerebellar kuru plaques. At the onset and early stages, patients manifested prominent, often mixed, cerebellar symptoms and memory loss, variably associated with behavioural/psychiatric and sleep disturbances. The cerebrospinal fluid prion real-time quaking-induced conversion assay was positive in 97.3% of cases, while 14-3-3 protein and total-tau positive tests were 52.6 and 75.9%. Brain diffusion-weighted MRI showed hyperintensity of the striatum, cerebral cortex and thalamus in 81.4, 49.3 and 33.8% of cases, and a typical profile in 92.2%. Mixed histotypes (MV2K + MV2-cortical) showed an abnormal cortical signal more frequently than the pure MV2K (64.7 versus 16.7%, P = 0.007). EEG revealed periodic sharp-wave complexes in only 8.7% of participants. These results further establish MV2K as the most common 'atypical' subtype of sporadic Creutzfeldt-Jakob disease, showing a clinical course that often challenges the early diagnosis. The plaque-type aggregation of the misfolded prion protein accounts for most of the atypical clinical features. Nonetheless, our data strongly suggest that the consistent use of the real-time quaking-induced conversion assay and brain diffusion-weighted MRI allows an accurate early clinical diagnosis in most patients.

Liver pathology in retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations: vasculopathic disease beyond nodular regenerative hyperplasia

Retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations (RVCL-S) is a rare autosomal dominant disease resulting from a frame-shift mutation in TREX1, an intracellular 3'-5' exonuclease 1. Hepatic findings include an elevated alkaline phosphatase (ALP) and nodular regenerative hyperplasia (NRH). Affected individuals typically succumb to brain lesions before clinically apparent hepatic manifestations; thus, little else is known about the hepatic pathology. Autopsy reports and a liver section from each (n=11) of three unrelated kindreds with the most common mutation in TREX1 (V235Gfs∗6) were studied with standard and immunohistochemical stains. Cases were compared with "normal liver" controls from similar autopsy years. Cases consisted of six men and five women who died at a median age of 50yr(range, 41-60 yr.). Seven had elevated ALP. Two had liver atrophy. Foci of NRH were variably detected in all. Inhomogeneous distribution of other findings included patternless parenchymal fibrous bands, approximation of vascular structures, and commonly, architectural changes of vascular structures. Only bile duct epithelia were unaffected. In addition, small trichrome-positive nodules were found along vein walls or isolated in the parenchyma. Rare foci of non-NRH hepatocytic nodules were noted in 3. Increased CD34 and altered α-SMA IHC expression were variably noted. Periportal ductules and perivenular K7 IHC expression were increased to unpredictable degrees. The extensive but inhomogeneous histopathologic findings in livers of autopsied patients with RVCL-S appear to involve hepatic vascular structures. These findings validate inclusion of vascular liver involvement beyond NRH in this complex hereditary disorder.

A20 as a Potential New Tool in Predicting Recurrence and Patient's Survival in Oral Squamous Cell Carcinoma.

A20, known as a potent inhibitor of NF-κB signaling, has been characterized in numerous clinical as well as preclinical studies. Recently, especially in various malignant diseases, the prognostic and therapeutic relevance of A20 was investigated. In oral squamous cell carcinoma (OSCC) however, the characterization of A20 is uncharted territory. We analyzed a tissue microarray (TMA) of 229 surgically-treated OSCC patients (2003-2013). Immunohistochemical (IHC) stainings were performed for A20 and CD3; additionally, standard haematoxylin-eosin staining was applied. IHC findings were correlated with a comprehensive dataset, comprising clinical and pathohistological information. A20 expression was analyzed in tumor cells as well as in tumor infiltrating lymphocytes (TILs) and correlated with the overall survival (OS) and recurrence-free survival (RFS) using uni- and multivariable Cox regression. The median follow-up time was 10.9 years and the A20 expression was significantly decreased in CD3+ TILs compared to mucosa-infiltrating lymphocytes (MILs). In the Kaplan-Meier analyses, higher A20 expression in TILs was correlated with better OS (p = 0.017) and RFS (p = 0.020). In the multivariable survival analysis, A20 overexpression correlated with improved OS (HR: 0.582; 95% CI 0.388-0.873, p = 0.009) and RFS (HR 0.605; 95% CI 0.411-0.889, p = 0.011). Our results indicate a novel prognostic role for A20 in OSCC. Due to its elevated expression in TILs, further research is highly desirable, which therefore could offer new therapeutic opportunities for patients suffering from OSCC.

Spectrally Selective Time-Resolved Emission through Fourier-Filtering (STEF).

We demonstrate a method for separating and resolving the dynamics of multiple emitters without the use of conventional filters. By directing the photon emission through a fixed path-length imbalanced Mach-Zehnder interferometer, we interferometrically cancel (or enhance) certain spectral signatures corresponding to one emissive species. Our approach, Spectrally selective Time-resolved Emission through Fourier-filtering (STEF), leverages the detection and subtraction of both outputs of a tuned Mach-Zehnder interferometer, which can be combined with time-correlated single photon counting (TCSPC) or confocal imaging to demix multiple emitter signatures. We develop a procedure to calibrate out imperfections in Mach-Zehnder interferometry schemes. Additionally, we demonstrate the range and utility of STEF by performing the following procedures with one measurement: (1) filtering out laser scatter from a sample, (2) separating and measuring a fluorescence lifetime from a binary chromophore mixture with overlapped emission spectra, (3) confocally imaging and separately resolving the standard fluorescent stains in bovine pulmonary endothelial cells and nearly overlapping fluorescent stains on RAW 264.7 cells. This form of spectral balancing can allow for robust and tunable signal sorting.

Dissection, Fixation, and Standard Staining of Adult Drosophila Ovaries.

Studies of the Drosophila ovary have provided significant insight into the molecular and cellular processes that control cell function, tissue organization, and animal development. To characterize mutants with defects in oogenesis or to observe the distribution of gene products involved in egg production, the ovaries must be carefully extracted and prepared for analysis. This chapter describes the manual dissection of ovaries from adult Drosophila females, followed by standard fixation and staining of the isolated tissue. Specifically, this chapter provides procedures for simple DNA and F-actin staining to assess cell and tissue morphology, as well as immunostaining to localize proteins of interest.

MORPHOLOGICAL FEATURES OF MAXILLARY SINUS CYSTS BASED ON THE STUDY OF PARTICULAR HISTOCHEMICAL AND IMMUNOHISTOCHEMICAL FACTORS OF INFLAMMATION.

The aim: To study the morphological structure and immunohistochemical markers of inflammatory cells in the maxillary sinus cyst wall. Materials and methods: Biopsy samples of maxillary sinus cysts of 92 operated patients aged 18 to 74 years were studied. We used standard staining with hematoxylin and eosin, PAS reaction, and immunohistochemical analysis with monoclonal antibodies to CD68 (macrophages) and CD3 (T- lymphocytes) to determine morphological features and severity of inflammation and PanCK AE1/AE2 antibodies to identify epithelium state. Results: All patients were divided into 2 groups: 29 patients with retention cysts and 63 with lymphagiotatic cysts (pseudocysts), depending on the unilateral or bilateral epithelial lining of the cyst wall. Retention cysts had the bilateral epithelial lining with a saving its function and structure. The thinned connective tissue layer was filled with separate clusters of lymphocytes and macrophages. Lymphagiotatic cysts were characterized by the unilateral epithelium lining of the wall with signs of degeneration, desquamation, and loss of the mucus-forming function. Dense infiltration of the connective tissue layer by inflammatory cells with pronounced expression is observed. Conclusions: Immunohistochemical analysis with monoclonal antibodies to CD3 and CD68 demonstrated that the inflammatory infiltrate of pseudocysts is represented by T-lymphocytes and different sizes of macrophages. The inflammatory infiltration of the pseudocyst wall was significantly severe compared to retention cysts.

ТИНКТОРИАЛЬНЫЕ И МОРФОЛОГИЧЕСКИЕ ХАРАКТЕРИСТИКИ МИКРОБНЫХ ОБЪЕКТОВ ДЛЯ ФОРМИРОВАНИЯ ЭЛЕКТРОННОГО АТЛАСА В СИСТЕМЕ АВТОМАТИЗИРОВАННОГО АНАЛИЗА

The authors present the results of automated analysis of gram positive and gram negative cocci and bacilli (Staphylococcus aureus, Neisseria spp., Corynebacterium spp., Escherichia coli) for a database that facilitates detection of changes in normal microflora and diagnosis of disbiotic conditions of various human biotopes. Following the standard sample preparations and Gram staining, microbial smears were investigated using an automated and computerized analyzer developed by the Mintz Radio Engineering Institute in Moscow. Morphological and tinctorial analysis established significant differences in cell area, perimeter and limit. Significant differences in tinctorial properties of microbial cells make possible image research using the RGB and HSV color spaces. Automated analysis enables identification of microbial cells simultaneously by digital images of morphological and color/brightness parameters. The proposed techniques can be helpful in constructing an electronic microflora atlas of various human biotopes.