Inorganic mercury (Hg) and methylmercury (MeHg) have emerged as global pollutants owing to their long-term environmental stability and bioaccumulation. These heavy metals enter aquatic systems via industrial emissions, coal combustion, and natural processes, posing a serious threat to ecosystems and human health. This study assesses the impact of Hg and MeHg on the growth and development of brine shrimp () nauplii by analyzing the histopathological effects on their tissues. Brine shrimp nauplii from the Bohai Bay in China were selected as the study subjects and exposed to 1 μmol/L solutions of HgCl₂ and MeHgCl. Tissue sections were continuously taken at different immersion times. After staining with the standard hematoxylin-eosin (HE) method, the tissue morphology of brine shrimp nauplii under different forms of Hg stress was observed under a light microscope. The results showed that MeHg exhibited significantly greater toxicity to brine shrimp nauplii than Hg. Under the same exposure time, the MeHg group exhibited more pronounced epithelial cell damage, nuclear material disorder, and nucleoplasm diffusion outside the nucleus than the HgCl₂ group. This finding provides an important theoretical support for further research into the toxicological mechanisms of MeHg and Hg, and highlights that the toxic effects of methylmercury on aquatic organisms.

Amyloidosis encompasses a spectrum of rare disorders characterized by extracellular amyloid deposition. Achieving an accurate early diagnosis of systemic amyloidosis necessitates biopsy-specific pathological evaluation. Formalin-fixed, paraffin-embedded liver biopsy specimens were examined using Congo red staining, electron microscopy, immunohistochemistry (IHC), immunofluorescence, and Congo red-assisted laser microdissection with mass spectrometry (LMD/MS). Failure Mode and Effects Analysis (FMEA) was employed for risk mitigation and quality control. Classical Congo red staining exhibited brick-red coloration, enhanced alkalinization, reduced permanganate staining, and characteristic apple-green birefringence under polarized light. Trypsin-digested IHC demonstrated kappa light chain positivity and lambda negativity, with improved background clarity compared to other retrieval methods-results concordant with electron microscopic colloidal gold staining, albeit with higher tissue consumption. Congo red-polarized microscopy permitted direct amyloid deposit localization. Subsequent LMD/MS identified immunoglobulin kappa light chain as the pathogenic precursor protein, though at increased expense. Congo red staining under polarized light remains the cornerstone technique for amyloid detection. LMD/MS provides superior specificity in amyloid typing relative to IHC, immunofluorescence, or electron microscopy, proving particularly advantageous for limited samples. Traditional methods remain valuable for validation when tissue is abundant. Histopathological assessment continues to be the diagnostic gold standard for hepatic amyloidosis; systematic integration and analytical refinement of these techniques are imperative for enhancing diagnostic accuracy.

Decalcification is an important step in histology laboratories to allow mineralized tissue samples to be trimmed and sectioned easily. Many decalcifying solutions have a rapid onset of action in softening tissues but alter protein structure and morphology, while others preserve protein integrity but are less efficient. The ideal decalcification protocol allows for rapid and cost-effective processing and precise evaluation of microscopy and antigen-based immunostaining such as immunohistochemistry. In our study, mouse tissues were decalcified with three commercially available solutions to identify the product that best meets those criteria. Immunocal (StatLab), Epredia, and Rapid-Cal (StatLab) decalcification agents were tested on formalin-fixed, paraffin-embedded CD-1 mouse femur, skull, and sternum samples. Multiple metrics including ammonium oxalate turbidity and radiography were used to assess stages of bone demineralization. Tissues were routinely processed, embedded in paraffin, and sectioned at 5 µm for H&E staining and CD3, CD31, and Iba1 immunostaining. Each tested sample represented a decalcification product and time (4, 6, 12, 24, 30, 48, 73 h). Samples were assessed by radiolucency on X-ray and gross bone pliability prior to histologic processing, followed by histopathologic scoring for completeness of demineralization, preservation of tissue architecture, and antigenicity of tissue. All three commercially available decalcifying solutions are sufficient for rapid decalcification with preservation of tissue integrity, cellular detail, and immunogenicity.

Placentas are temporary organs needed to support a developing embryo and arise from both embryonic and maternal tissues. Calcifications of tissues outside of bone and teeth mineralization are often a sign of tissue damage and impaired organ function. Placental calcifications have been described previously in the literature and usually increase in normal pregnancies as the placenta ages, but they have also been associated with the potential for fetal distress. This study utilized 139 placental tissues from singleton control placentas (51), fused twin placentas (48), and non-fused twin placentas (40) with weights over 320 grams (third trimester) and similar maternal ages to determine which placental type(s) exhibited the largest density of calcifications using the von Kossa stain for calcium salts. The study found there were no differences in calcification densities among placenta types, suggesting that twin pregnancies do not experience additional placental stress risk from calcifications. Importantly, the finding that nearly all third-trimester placentas contained calcifications when systematically evaluated indicates that calcification may represent a normal maturational process rather than a pathologic sign of distress. These results help to caution the interpretation of placental calcifications and may provide reassurance to patients and providers managing twin pregnancies.

Toxicologic pathology is undergoing a digital transformation, with advances in imaging and computational methods enabling automation of traditionally manual workflows. Central to these digital workflows is the generation of high-quality whole slide images (WSIs), where one key determinant of image quality is focus sharpness. To address this, we have integrated a pair of productionalized computational models - 'MiQC' (Microscopic Quality Control) - into our routine image QC workflows. MiQC combines Local Binary Patterns (LBP) and DeepFocus-based deep learning algorithms to detect and quantify out-of-focus regions in WSIs. Subsequent to scanner-based focus metric assessment, MiQC further screens WSIs and supports technician review by generating heatmaps that highlight problematic areas. Even WSIs with scanner focus scores of 98-99% can contain unacceptable blur, which MiQC helps identify. Using this system, 85-95% of WSIs are approved without further intervention, and technician review time is reduced by nearly 50%. Compared to fully manual review, MiQC has doubled our throughput of QC'd slides per hour. This efficiency gain has accelerated the expansion of our high-quality WSI repository and provides a scalable, reproducible framework for enhancing image QC in toxicologic and broader digital pathology applications. MiQC supports higher throughput and integration of automated image analysis pipelines, laying the groundwork for robust downstream computational pathology workflows.

Glial fibrillary acidic protein (GFAP) immunohistochemistry staining is specific to glial cell intermediate filaments. GFAP has been reported in some diseases related to injury of central nervous system. The actual normal biological function of GFAP in the retinas of some vertebrates is still under investigation. The current study examines the distribution of GFAP in the retinas of various species of amphibian, reptilian, birds, and mammals by immunohistochemical technique. The results indicated that GFAP localization in the retina is related to specific species. Unique, homogeneous GFAP labelling was observed in retina, which was different from other studied reptiles (, ). Low GFAP labelling was noted in the retinas of birds and some species of the studied mammals (, , and ). However, other studied mammals () showed different intensities of GFAP expression. The study finds that GFAP helps to clarify the potential function of Müller cells in the regeneration process in different species.

The retinal pigment epithelium (RPE) is associated with the emergence and development of diabetic retinopathy. Interestingly, a previous clinical study observed that the atrophy of RPE cells surrounding the optic disc in type 1 diabetic children were significantly less pronounced compared to normal children, contradicting current reports. In order to explore the molecular mechanisms behind this contradictory phenomenon, we conducted a series of experiments and reached the following results. First, RPE cells proliferation increased in a glucose concentration-dependent manner , accompanied by elevated Brachyury and CTGF protein expression, but decreased overall cell viability. Secondly, experiments and diabetes mouse models confirmed that insulin promoted RPE cell proliferation in high glucose concentrations by activating ERK1/2 phosphorylation. Furthermore, insulin down-regulated the expression of Brachyury and CTGF proteins, possibly reducing high-glucose‒induced damage to RPE cells. In conclusion, the effect of insulin treatment on the proliferation of RPE cells was significantly more significant than that of hyperglycemia, which may be related to the activation of Erk1/2 or reduction of RPE cell damage by inhibiting the occurrence of EMT.

With an increasing concentration of microplastics (MPs) in every biome, laboratories with a focus on creating histology slides from resin-embedded specimens could be partially responsible for expanding the emission of microscopic resinous particles into the environment. With current research elucidating harmful health impacts from MPs, releasing them incautiously is arguably unethical and, in the near future, plausibly illegal. The Orthopedic Bioengineering Research Laboratory (OBRL) is in Colorado, a state known not only for its natural beauty but also for its increasing number of legislative amendments aimed at reducing plastic pollution. The histology department of the OBRL has chosen to self-regulate due to the importance of protecting health and the environment. Because virtually every molecule of plastic ever created is still in existence, a greater need for MP research and mitigation has become apparent. Remediation is specifically important due to findings indicating negative impacts on neurodevelopment, neuronal organelle function, mental health, and the increased risk of dementia.

Due to the prevalence of hematoxylin and eosin (H&E) staining in routine histological preparations, understanding the factors that impact stain color characteristics is vital to attain consistently high-quality stains. In the last decade, increased use of digital pathology and image analysis (specifically by optical density [OD] measures) has provided new ways of assessing staining precision. This paper combines data from two studies that tracked H&E staining quality in both nuclear and cytoplasmic components of 12 tissues (11 human and 1 porcine) by OD after overuse of H&E staining reagents from 5 vendors. Both studies showed a decrease in eosin stain intensity by OD and visual inspection (by a histologist) with reagent overuse. This trend varied in degree by tissue type and reagent vendor. Nonetheless, staining quality for H&E staining from all vendors and for all organs remained acceptable (but not always optimal) for microscopic evaluation by the College of American Pathologists (CAP) and National Society for Histotechnology (NSH) staining criteria when stained sections were reviewed by a board-certified veterinary pathologist.

Build-up of static electricity frequently hampers proper production and handling of serial paraffin sections produced on a microtome, a problem that is exacerbated in modern, fully climatized laboratories. In this context, we review a number of suggested remedies for static electricity, concerning increase of humidity, in one way or the other, and various kinds of anti-static devices. An excellent solution, the shockless one-point ionizing bar that we have implemented in our laboratory, albeit not new, is presented here in detail.

There are several variations of laboratory procedures to preserve and demonstrate monosodium urate (MSU) crystals in tissue for the diagnosis of gout. MSU is water soluble and washed away in most staining solutions, so these procedures generally necessitate two slides; one which is silver stained and one which is left unstained to confirm the negative birefringence of MSU when viewed under polarized light. The modified Gomori's methenamine silver (GMS) for urates is an underutilized procedure which provides sensitive, high-contrast staining of MSU crystals. Additionally, we have observed that MSU deposits are preserved in the stained sections, allowing for examination by polarized light and subsequent diagnosis with only one slide. We have found this modification of the GMS stain to be the most sensitive and efficient method for the diagnosis of gout histologically due to the procedure's relatively short duration as well as the sharp black staining of MSU crystals and preservation of MSU birefringence on a single slide.

Histological preparation paraffin embedding is the gold standard method for evaluating tissue structure and composition, whether it is originated from biopsy or engineered . Quite often, deformation and shrinkage occur during the histological preparation, which are difficult to predict and qualify. The present study investigates the morphometric changes in bioprinted hydrogels composed of alginate and gelatine, common tissue engineering materials, focusing on three morphologies: full slabs, porous slabs, and porous cubes. These structures underwent key histological steps, including fixation, processing (dehydration, clearing, and infiltration with melted paraffin), embedding, and slicing, to evaluate their shrinkage behavior. Shrinking factors were systematically measured, showing that processing had the most significant effect (34-40% shrinking), followed by fixation (20-28% shrinking). Porous structures exhibited greater shrinkage compared to full slabs due to their internal geometry. Additionally, anisotropic behavior was observed in porous cubes, with different shrinking factors in the XY plane (horizontal) and Z direction (vertical), leading to an overall volumetric shrinking factor of 81.3%. The results demonstrated the critical influence of hydrogel structure on deformation and emphasized the need for tailored histological protocols to maintain structural fidelity. While this study focused on hydrogels alone, future work will incorporate cellularized bioengineered tissues to evaluate the impact of cell-mediated remodeling and extracellular matrix deposition on histological outcomes. This research offers a framework for optimizing histological preparation in bioengineered tissues, enabling more accurate assessment of their structure and function for regenerative medicine applications.

The claustrum is a sheet-like layer of gray matter situated between the external and extreme capsules of the mammalian brain. This structure was first described by the French physician and anatomist Vicq d'Azyr in 1786. The claustrum's phylogeny, ontogeny and functional characteristics have long been the subject of debate and considerable investigative efforts. However, despite such efforts (or perhaps as a result thereof), significant disparities and discrepancies remain, most notably in the context of the claustrum's afferent and efferent connections. For the purpose of this study, we sought to focus our efforts on fronto-claustral and occipito-claustral connections. Twelve healthy, adult male cats, each weighing ~ 3.5 kg, were studied, seven of which underwent electrolytic lesions of the frontal cortex (A3, A4, and a portion of A6), and five of the occipital cortex (A17, A18, A21). From three to six days after lesioning, subjects were euthanized in accordance with ethical norms. After the brains were removed and blocked, samples of the claustrum were taken and prepared for electron microscopy. Three to six days after lesions of the frontal cortex, we observed an abundance of degenerative boutons in the dorsal claustrum. The vast majority of boutons exhibited the characteristic signs of dark degeneration, whereas only 10% appeared to have undergone light degeneration. Similar results were seen in the dorsal claustrum over the same period of time following lesions of the visual cortex. These results suggest that the dorsal claustrum receives at least two types of connections - separately and concurrently - from the frontal and occipital cortices.

Bladder cancer diagnosis is challenged by invasive monitoring and workflow inefficiencies impacting diagnostic reliability. This prospective study enrolled 150 patients (January 2020-December 2022) and evaluated a novel liquid-based immunocytochemistry platform, coupled with integrated machine learning, for detecting urothelial carcinoma in voided urine. All 150 cytology slides met the adequacy threshold of ≥2,644 urothelial cells and showed preserved cytomorphology. Eight cases of papillary urothelial neoplasm of low malignant potential (PUNLMP) were set aside a-priori, yielding an analytic cohort of 142 patients (115 urothelial-carcinoma, 27 benign) for performance analysis. hTERT (sensitivity 92.2%, specificity 66.7%), GATA-3 (67.0%, 88.9%), and CK17 (89.6%, 66.7%). In multi-marker analysis, sensitivity reached 100% (95% CI 96.8-100) when any marker was positive, whereas specificity reached 100% (95% CI 87.3-100) when all three markers were positive. The workflow-optimized platform standardizes specimen preparation and multi-marker interpretation, offering a robust foundation for urine-based bladder-cancer diagnostics. Larger, multi-center validation studies are warranted to refine specificity estimates and facilitate laboratory integration. This study demonstrates that addressing fundamental workflow challenges in bladder cancer diagnostics before implementing artificial intelligence creates more effective diagnostic tools. By prioritizing specimen integrity and standardization through a novel liquid immunocytochemistry platform, exceptional diagnostic performance was achieved with 100% sensitivity and specificity under defined marker parameters across various cancer stages. This workflow-first approach to integrating machine learning with advanced biomarker analysis offers a model for developing clinically practical diagnostic innovations that can reduce reliance on invasive monitoring procedures while improving detection accuracy.

The incidence of lymphoma, a cancer that affects both humans and animals, has witnessed a significant increase. In response, immunohistochemistry (IHC) has become an essential tool for its classification. This prompted us to develop an innovative mathematical methodology for the precise quantification of immunopositive and immunonegative cells, along with their spatial analysis, in CD3-stained lymphoma IHC images. Our approach involves integrating an algorithm based on a mathematical color model for cell differentiation, employing the distinctive morphological erosion, algorithmic transformations, and customized histogram equalization to enhance features. Refined local thresholding enhances classification precision. Additionally, a customized circular Hough transform quantifies cell counts and assesses their spatial data. The algorithms accurately enumerate cell types, reducing human intervention and providing total numbers and spatial information on detected cells within tissue specimens. Evaluation of IHC image samples revealed an overall accuracy of 93.98% for automatic cell counts. The automatic counts and location information were cross-validated by three pathology specialists, highlighting the effectiveness and reliability of our automated approach. Our innovative framework enhances lymphoma cell counting accuracy in IHC images by combining physics-based color understanding with machine learning, thereby improving diagnosis and reducing the risks of human error.

Formalin-fixed paraffin embedded tissues are a mainstay in contemporary histology and pathology practice and represent the most readily available and widely used sample format that has guided early studies and set standards for a new field of spatial biology. However, fixed tissues are not always suitable for all molecular applications and detection of certain molecular targets can be suppressed by these practices. There is a greater diversity of analysis techniques that are commercially available to researchers today, and pathologists and core services are now engaging with groups that prefer fresh-frozen samples more than ever before. Some of these analysis techniques can be characterized as 'label-free' and access unique classes of molecular targets in situ. Our team has developed tools and procedures to facilitate a synergy between the realm of label-free spatial biology and the histology laboratory for biomedical discovery programs. Herein, we discuss the development of 3D printed cassette holders for use in a variety of commercially available cryostats. This apparatus was tested using fresh frozen tissue slices embedded in hydroxypropyl methylcellulose polyvinylpyrrolidone for subsequent analyses. Based on our tests, we propose this protocol as an option to better preserve sample history during the lifetime of a specimen and curtail the decomposition of any lower abundance endogenous chemical moieties for spatial 'omics' analysis using mass spectrometry.

Bone tissue poses critical roadblocks for spatial transcriptomics and molecular pathology due to a combination of its dense, calcified matrix and inadequate preservation of biomolecules in conventional decalcification. Decalcification is a complex and nuanced histological process to concomitantly preserve nucleic acids, proteins, and tissue architecture, ensuring molecular integrity for downstream assays. However, commonly used agents like formic and hydrochloric acids, while efficient, can degrade biomolecules to varying extents, complicating assays such as PCR, sequencing, immunohistochemistry, and hybridization. Advances in spatial transcriptomics, both sequencing- and imaging-based, emphasize the importance of optimizing decalcification protocols to improve research outcomes. This synoptic and perspective article explores traditional and modern decalcification methods, offering recommendations on technical and methodological refinements for achieving molecularly robust processing of bone and calcified tissues in spatial transcriptomics and molecular pathology.