Journal of Histotechnology最新文献

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 in situ 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.

The application of Clear Gorilla Glue® household adhesive to microscope slides has been found to enhance the mounting and retention of traditionally difficult tissue types, notably bone and tooth specimens. Improvement in end results were observed across H&E staining, immunohistochemistry, and in situ hybridization.

Gomori methenamine silver (GMS) stains are commonly utilized to exclude fungal esophagitis in esophageal biopsies. Our laboratory protocol for special stains, including GMS, stipulates that two levels be performed for each stain. We retrospectively reviewed 127 esophageal biopsies which had GMS stains performed; there were 62 GMS-positive and 65 GMS-negative cases. Sixty-one of the positive cases (98%) had fungal organisms present on all levels. Only one case showed fungal elements on the first, but not the second, level. Given national laboratory staff shortages, performing levels for special stains may be unnecessary, inefficient, and time consuming in the surgical pathology laboratory.

H vessels are an essential link in angiogenic-osteogenic coupling and orchestrate the process of bone healing. H vessels are critically deficient in the setting of radiation-induced fractures, which have been reported to occur in up to 25% of patients undergoing radiotherapy. By increasing H-vessel proliferation, Deferoxamine (DFO) revitalizes the physiologic response to skeletal injury and accelerates irradiated fracture repair. H-vessel quantification is therefore an important outcome measure in histologic analysis of bone healing. However, an optimized protocol for staining H vessels in formalin-fixed paraffin-embedded (FFPE) tissue sections has not been reported. With this protocol, we describe a method of staining FFPE bone samples with minimal background fluorescence and high signal-to-noise ratio. We examined mandibular specimens in a rat model of bone healing from a range of fracture conditions, including healthy bone (Fx), irradiated bone (XFx), and irradiated bone with DFO treatment (XFx-DFO). Quantitative analysis revealed a significant increase of H vessels in the XFxDFO group compared to both the Fx and XFx groups. By optimizing immunofluorescent staining of H vessels in FFPE samples across a range of fracture conditions, we offer investigators an efficacious means of producing reliable imaging for quantitative analysis of H vessels in an irradiated fracture callus.

Stabilized hyaluronic acid (HA), produced through diverse cross-linking technology and formulated as an injectable gel, has found widespread utilization in aesthetic industry. Cross-linked HA essentially constitutes a gel particle composition formed by numerous viscoelastic particulates. Various product formulations yield HA gels with distinct properties, including particle size, viscoelasticity, and interaction forces between particles. While previous studies have primarily concentrated on the biological safety and macroscopic expression of fillers, limited research exists on the internal mechanisms governing their macro-performance. This study selected three common dermal fillers for analysis, establishing an animal model to assess their in vivo interaction with surrounding tissues and explore their internal mechanisms. The findings revealed that particle size plays a crucial role in tissue integration.

Age determination of bottlenose dolphins (Tursiops truncatus) is a critical tool in understanding both individual and population health. There are many methods of aging bottlenose dolphins including analysis of teeth, pectoral flipper radiographs, and epigenetics. The most common and oldest method for aging toothed cetaceans is the counting of growth layer groups (GLGs) in the teeth. Current techniques have technical and repeatability challenges. Therefore, a processing technique that results in better resolution of GLGs is needed. This study compares different decalcifications and different histochemical staining techniques. Decalcification was done using 10% EDTA, Kristensen's decalcification, and Rapid Decalcification Solution (RDO). Following decalcification and routine processing, GLGs were assessed using Hematoxylin and Eosin (H&E), hematoxylin, Giemsa, Wright-Giemsa, Toluidine Blue (T-Blue), Masson's Trichrome, and Congo Red staining techniques. Decalcification with Kristensen's and staining with Masson's Trichrome and Congo Red were determined to best highlight GLGs. This processing and staining was then applied to a sample population of 102 bottlenose dolphins that were evaluated independently and blindly by two observers. Of the 102 dolphin samples, 13 (12.7%) were unable to age due to no clear distinction or distortion between GLGs.

The anaphylatoxin C5a and its receptor C5aR (CD88) are complement pathway effectors implicated in renal diseases, including ANCA-associated vasculitis. We investigated the kidney expression of C5aR and a second C5a receptor C5L2 by using immunohistochemistry, in situ hybridization, and spatial gene expression on formalin-fixed, paraffin-embedded human and mouse kidney. C5aR was detected on interstitial macrophages and in multiple tubular regions, including distal and proximal; C5L2 had a similar expression pattern. The 5/6 nephrectomy model of chronic kidney injury exhibited increased C5aR expression by infiltrating cells within the fibrotic regions. C5aR expression was confirmed on human leukocytes and in vitro differentiated macrophages by flow cytometry, and treatment with C5a induced the expression of chemokines and remodeling factors by macrophages, including CCL-3/-4/-7, -20, MMP-1/-3/-8/-12, and F3, and promoted leukocyte survival. C5a activity was C5aR dependent, as demonstrated by reversal with the C5aR inhibitor avacopan. Collectively, these results suggest that myeloid C5aR may induce excessive inflammation in the kidney via immune cell recruitment, extracellular matrix destruction, and remodeling, resulting in fibrotic tissue deposition.

High glucose-induced dysfunction of endothelial cells is a critical and initiating factor in the genesis of diabetic vascular complications. Low-magnitude high-frequency vibration (LMHFV) is a non-invasive biophysical intervention. It has been reported that it exhibits protective effects on high glucose-induced osteoblast dysfunction, but little was known on diabetic vascular complications. In this work, we aim to clarify the role of LMHFV on high glucose-induced endothelial dysfunction and hypothesized that the protective effects functioned through adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway. We cultured primary murine aortic endothelial cells (MAECs) in normal or HG medium, respectively, before exposing to LMHFV. The tube formation, paracellular permeability assay, and aortic ring sprouting assay showed that the high glucose injured-function of MAECs was improved after LMHFV treatment. The intracellular ROS generation analysis, mitochondrial complex I activities measurement, ATP measurement and mitochondrial membrane potential (MMP), and mitochondrial ROS generation analysis of MAECs indicated that mitochondrial function was restored by LMHFV loading in a high glucose environment. Mechanically, western blot assays showed that AMPK phosphorylation was promoted and mTOR was inhibited in LMHFV-induced endothelial function restoration. After the administration of the AMPK inhibitor, Compound C, these protective effects resulting from LMHFV are reversed. These findings suggest that LMHFV plays a significant role in protecting endothelial cells' function and mitochondrial function in high glucose-induced injured MAECs via AMPK/mTOR signalling.

Stains frequently performed to exclude infectious etiologies in esophagitis include Grocott methenamine silver (GMS) and periodic acid-Schiff (PAS) as well as immunohistochemistry (IHC) assays for cytomegalovirus (CMV) and herpes simplex virus (HSV). The diagnostic yield of these tests, in this situation, has not been well studied. We retrospectively reviewed 261 esophageal biopsies, which had one or more of the above tests performed. The diagnostic yield for GMS and PAS was 8%, while CMV and HSV immunohistochemistry had a diagnostic yield of 1% and 0%, respectively. Our study suggests that routine use of ancillary labeling techniques in esophagitis biopsies may be of limited utility and have low diagnostic yield.