Methods and Protocols最新文献

{"title":"Method and Analysis of High-Calcium Induced Differentiation of Human Primary Keratinocytes <i>In Vitro</i>.","authors":"Sarmistha Mahanty, Dharna Saini","doi":"10.3390/mps9050126","DOIUrl":"10.3390/mps9050126","url":null,"abstract":"<p><p>Epidermal development and regeneration rely on the stepwise differentiation of keratinocytes, the primary constituent cells of the epidermis. In the integrated epidermis, keratinocyte differentiation (KD) occurs in response to the increasing calcium gradient. Reflecting this, high-calcium incubation remains a popular approach for inducing KD <i>in vitro</i>. The outcome is primarily assessed by differential expression of differentiation markers compared to proliferative cells. However, it is significantly influenced by the cell models used, calcium concentration and incubation time, which often vary between studies. Such differences compromise reproducibility and comparability. In this article, we overview major parameters that shape the outcome of high-calcium induced KD. Comparative experimental data between high-calcium incubation and high-density induced differentiation underscore the need for combining additional parameters, such as microscopy based visualization with marker protein analysis for unbiased assessment of KD. These recommendations are intended to guide future research in keratinocyte biology.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 5","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13600111/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149030945","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of Two Methods for Decellularizing Human Amniotic Membranes for Use in Tissue Engineering.","authors":"Elga Johanna Vargas, Carlos Domínguez-Paz, Lina Andrea Gómez","doi":"10.3390/mps9050127","DOIUrl":"10.3390/mps9050127","url":null,"abstract":"<p><strong>Background: </strong>The human amniotic membrane (hAM) is widely used in tissue engineering due to its low immunogenicity and extracellular matrix (ECM) rich in structural proteins; in this context, decellularization aims to remove cellular components while preserving tissue integrity, and this study aimed to compare the effects of two enzymatic methods (trypsin and thermolysin).</p><p><strong>Methods: </strong>Human amniotic membranes obtained from placentas were divided into three groups: non-decellularized control, trypsin-treated, and thermolysin-treated. The membranes were treated with 0.25 trypsin-EDTA for 60 min at 37 °C, followed by incubation with Triton X-100 for 30 min at 37 °C, or with 125 µg/mL thermolysin for 9 min at 37 °C. Histological analysis with hematoxylin-eosin and semi-quantitative scoring assessed epithelial and stromal integrity, immunohistochemistry evaluated laminin α and fibronectin expression with quantitative image analysis, and uniaxial tensile testing measured mechanical properties; no preregistration numbers or animal models were reported.</p><p><strong>Results: </strong>Trypsin achieved more effective epithelial removal, whereas thermolysin caused greater stromal disruption; no statistically significant differences in laminin α or fibronectin expression were found among groups (<i>p</i> > 0.05), indicating preservation of key ECM components; both treatments reduced mechanical performance, with thermolysin decreasing early- and mid-path stiffness and trypsin reducing late-path stress and work density.</p><p><strong>Conclusion: </strong>Both enzymatic protocols preserved ECM composition but induced distinct structural and mechanical alterations, suggesting that method selection should balance efficient cell removal with preservation of structural and functional properties according to the intended application.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 5","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13600013/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149030900","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Practical Workflow for Correcting Kit-Specific Effects in Whole-Exome Sequencing Data.","authors":"Laura Jarosz, Marcel Ochocki, Julia Merta, Lajos Pusztai, Michal Marczyk","doi":"10.3390/mps9050125","DOIUrl":"10.3390/mps9050125","url":null,"abstract":"<p><p>Large-scale, multi-center projects have become common in the era of rapid technological development, but protocol standardization remains challenging. In whole-exome sequencing (WES), various exome enrichment kits exhibit variable efficiency across genomic regions, leading to systematic, non-biological batch effects, much stronger than other technical factors. We propose a workflow to minimize the effect of WES capture inconsistencies in single-nucleotide variation (SNV) data. The pipeline consists of quality control, mapping to the genome, SNV calling, joint genotyping, and imputing genotypes using reference haplotypes. SNVs are then aggregated into gene-level features measuring the burden of deleterious variants. Finally, a gene-level imputation is performed using a customized algorithm. Namely, if the detection rate of a gene is low in samples enriched with a given capture kit but high in samples enriched with other kits, missing values in the former group are imputed, as such differences are unlikely to reflect true biology. As a benchmark, we conducted a study on over a thousand breast cancer cases across 11 cohorts, using eight exome capture kits. We demonstrated that the proposed pipeline leads to a considerable decrease in the batch effect signal, potentially increasing the likelihood of finding true biological signals.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 5","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13600025/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149030894","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluating Cell-Free DNA Quantification and Integrity in Breast Cancer Using Fluorometric, Electrophoretic and PCR-Based Platforms.","authors":"Gisha Rose Antony, Vidya P Nimbalkar, Sowmya Nagaraj, Mahalakshmi Subramanya, Anupama C Eshwara Rao, Ganapathi Bantwal, Belinda George, Akshay Ajoy, Nihal M Ahamed, Rakesh S Ramesh, Jyothi D N, Jaya Kumari S, Jyothi S Prabhu","doi":"10.3390/mps9050124","DOIUrl":"10.3390/mps9050124","url":null,"abstract":"<p><p>Cell-free DNA (cfDNA) has emerged as a promising liquid biopsy biomarker; however, differences in analytical methods may influence cfDNA quantification and integrity assessment. This study compared plasma cfDNA concentration and integrity between treatment-naïve breast cancer (BC) patients (<i>n</i> = 100) and healthy controls (HC; <i>n</i> = 70) using complementary analytical platforms. cfDNA concentration was measured by Qubit fluorometry, TapeStation (TS) electrophoretic profiling, and ALU-based quantitative PCR (qPCR). An exploratory genomic DNA (gDNA) correction of Qubit-derived cfDNA concentration was performed using TS-derived fragment-size information. cfDNA integrity was assessed using the ALU-derived DNA Integrity Index (DII (ALU); ALU247/ALU115) and the TS-derived DNA Integrity Index (DII (TS)), and methods were compared for their correlation and agreement. Qubit- and TS-based cfDNA measurements were significantly higher in BC patients than in HC. Among the ALU-based qPCR measures, although ALU115 did not differ significantly between groups, ALU247 and DII (ALU) were significantly higher in BC patients, indicating an enrichment of longer cfDNA fragments. Exploratory gDNA correction did not substantially alter the observed differences. Significant positive correlations were observed among the cfDNA quantification methods. Supporting these findings, agreement analysis demonstrated improved agreement following gDNA correction. In contrast, cfDNA integrity assessment was method-dependent: although DII (ALU) was significantly higher in BC patients, DII (TS) did not differ significantly between groups, and no significant correlation or agreement was observed between the two indices, indicating that the two methods evaluate different lengths of cfDNA fragments. These findings demonstrate that complementary analytical platforms consistently differentiated BC patients from HC based on cfDNA concentration, whereas cfDNA integrity assessment depended on the analytical method employed. Together, these findings provide methodological insights into cfDNA quantification and fragmentation assessment and support the complementary use of fluorometric, electrophoretic, and ALU-based qPCR approaches for comprehensive cfDNA characterization.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 5","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13600121/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149030877","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparison of the Utility of Amplitude-Spectral and Coherence Features of Psychotropic Drugs' Action on ECoG Signal for Pharmaco-EEG Based Drug Screening in Rats.","authors":"Yuriy I Sysoev, Nikita S Kurmazov, Darya D Shitc, Sergey V Okovityi","doi":"10.3390/mps9050123","DOIUrl":"10.3390/mps9050123","url":null,"abstract":"&lt;p&gt;&lt;p&gt;A naive Bayesian classifier (NBC) combined with principal component analysis (PCA) effectively differentiates the dose-dependent effects of certain groups of psychoactive drugs based on their impact on the amplitude-spectral characteristics of electrocorticograms (ECoG) in rats. This approach has been shown to be useful for pharmacological screening of agents with unknown or poorly understood activity. Despite previously obtained optimistic results, classification determination for some drugs was inaccurate, necessitating the search for possible ways to improve the predictive effectiveness of the proposed algorithm. One possible approach would be to use as input quantitative data not only the impact of the psychoactive drugs studied on the amplitude-spectral characteristics of ECoG but also connectivity changes, including the average coherence power of different pairs of leads. &lt;b&gt;The aim&lt;/b&gt; of this study was to compare the accuracy of NBC in classifying the pharmacological mechanism of action of agents with well-known mechanisms (test set) using pharmaco-EEG data on changes in the amplitude-spectral characteristics of ECoG, coherence, and the combined use of two data sets. &lt;b&gt;Materials and methods.&lt;/b&gt; Experiments were performed on Wistar rats with chronically implanted ECoG electrodes. The training set, relative to which the effects of the pharmacological agents from the test set were classified, were the matrices of effects of 12 pharmacological agents: the NMDA antagonist dizocilpine, the D2/D3 antagonists haloperidol and sulpiride, the M-anticholinergic tropicamide, the H1/5HT2A receptor blocker hydroxyzine, the acetylcholinesterase inhibitor galantamine, the alpha-2 adrenergic agonist dexmedetomidine, the alpha-2 adrenergic antagonist atipamezole, the adenosine receptor blocker caffeine and the GABA-mimetics aminophenylbutyric acid (phenibut), bromdihydrochlorophenylbenzodiazepine (phenazepam) and 5-ethyl-5-phenyl-2,4,6(1H,3H,5H)-pyrimidinetrione. The test set included various drugs with tropism for the targets of the training set drugs: dopamine receptor antagonists chlorpromazine, droperidol, tiapride and raclopride, H1-histamine blockers diphenhydramine and promethazine, 5-HT2-receptor blockers ritanserin and glemenserin, acetylcholinesterase inhibitor ipidacrine, alpha2-adrenergic receptor antagonist yohimbine, alpha2-adrenergic agonists medetomidine and xylazine, GABA-mimetics 5-ethyl-5-(1-methylbutyl)-2,4,6(1H,3H,5H)-pyrimidinetrione and chloral hydrate. The analysis of the ECoG signal included the calculation of 132 amplitude-spectral characteristics and 75 coherence indicators, which, using the PCA, led to new integrative indicators used for further classification of the NBC. &lt;b&gt;Results and discussion.&lt;/b&gt; For each drug in the test set, the median similarity probability with a particular group from the training set was calculated, which was used to assess the classification quality. It was found that, when using the amplitude","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 5","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13600116/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149030947","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Suitability of a Fluorescence Method for Flavin Mononucleotide Determination in Modified HTK-Based Perfusion Solutions During Hypothermic Oxygenated Perfusion of Liver Grafts.","authors":"Yulia B Basok, Eugenia G Kuznetsova, Aleksandra D Belova, Dmitriy A Morozov, Mikhail A Boldyrev, Artem R Monakhov, Nikita V Grudinin, Vladimir K Bogdanov, Denis M Bondarenko, Stepan I Zubenko, Nidzhat M Yusuf, Sergey V Gautier","doi":"10.3390/mps9040122","DOIUrl":"10.3390/mps9040122","url":null,"abstract":"<p><p>A comprehensive assessment of allograft quality is essential for predicting successful transplantation and graft survival. Elevated levels of flavin mononucleotide (FMN) in perfusate obtained during hypothermic oxygenated perfusion (HOPE) may serve as a promising predictor of early graft dysfunction and post-transplant complications. The aim of this study was to evaluate the feasibility of applying an FMN determination method to HTK-based perfusion solutions used during liver machine perfusion. The spectrofluorimetric method for FMN determination in modified HTK perfusion solution (modHTK) was evaluated for the following parameters: linearity, accuracy, repeatability, reproducibility, and stability. The study demonstrated the suitability of the quantitative FMN determination method for modHTK solution used during HOPE of liver grafts.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13515777/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148830477","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimisation and Validation of a Microscale FRAP Assay with Matrix-Matched (Interaction-Corrected) Blanking for Accurate Antioxidant Capacity Assessment of Honeys.","authors":"Ivan Lozada Lawag, Sharmin Sultana, Lee Yong Lim, Cornelia Locher","doi":"10.3390/mps9040120","DOIUrl":"10.3390/mps9040120","url":null,"abstract":"<p><p>This study optimised and validated a microplate Ferric-Reducing Antioxidant Power (FRAP) assay for honey that incorporates a honey-specific matrix-matched blank-a paired absorbance measurement lacking the chromogenic probe 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ)-to separate true ferric reduction from non-reductive Fe³⁺-matrix interactions that otherwise inflate apparent antioxidant capacity. A 30 min incubation at 37 °C in the dark was selected as the optimal compromise between reaction completeness and reagent/matrix stability, using 20 µL sample and 180 µL FRAP reagent per well (absorbance at 620 nm). The assay was validated according to International Council for Harmonisation (ICH) principles, showing linearity for FeSO₄·7H₂O from 200-1200 µM (R² ≥ 0.9988), Limit of detection/limit of quantification (LOD/LOQ) of 0.28/0.94 mmol Fe²⁺ equivalents kg^-1, recoveries of 101-103%, and precision ≤ 5% relative standard deviation (RSD). All tested individual sugars except maltodextrin and the artificial honey matrix produced FRAP responses below the LOQ. Flavonoids, however, showed pronounced, time-dependent, non-reductive Fe³⁺ interactions and were markedly overestimated under conventional water blanking, while phenolic acids were less affected. Applied to 47 Western Australian honeys, the validated assay yielded FRAP activities of 2.80-9.52 mmol Fe²⁺ kg^-1; matrix-matched blanking gave consistently lower, more chemically specific values than conventional water blanking, with corrections ranging up to >30% depending on honey type. Matrix-matched blanking is therefore essential, not optional, for accurate FRAP measurement in honey, and the same principle should apply directly to other phenolic-rich foods like wine, tea, and fruit extracts-wherever iron-polyphenol interactions confound conventional antioxidant assays.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13515261/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148830523","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The IMAGE Statement: A Proposed Reporting Guideline for Clinical Images-A CARE Guideline Extension.","authors":"Howard Lopes Ribeiro Junior, Humberto Morais, Fidel Manuel Cáceres-Loriga, Mauer Alexandre da Ascensão Gonçalves","doi":"10.3390/mps9040121","DOIUrl":"10.3390/mps9040121","url":null,"abstract":"<p><p>Clinical image publications represent an important educational resource across healthcare disciplines, providing concise visual demonstrations of diseases, diagnostic findings, and therapeutic outcomes. Despite their widespread use, reporting practices remain highly heterogeneous, with substantial variability in the description of clinical context, image acquisition, ethical considerations, and educational content. Existing reporting guidelines, including the CARE Guidelines, do not specifically address the unique methodological and technical aspects of image-based publications. The aim of this study was to develop the IMAGE Statement (Improving Clinical Image Reporting Standards), a reporting guideline specifically designed for clinical image publications as an extension of the CARE Guidelines. The development process was informed by recommendations from the EQUATOR Network and included a literature review, analysis of journal instructions for authors, generation of candidate reporting items, and expert consensus. The resulting IMAGE Statement comprises 15 essential reporting items covering the title, patient information, clinical context, diagnostic assessment, image acquisition, image quality, image selection, image annotation, image description, diagnostic interpretation, final diagnosis, educational message, ethics and consent, artificial intelligence use disclosure, and figure legend. The IMAGE Statement provides a structured framework intended to improve completeness, transparency, reproducibility, educational value, and ethical reporting of clinical image publications across healthcare disciplines.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13515853/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148830492","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Wheel Diameter Affects Vibration Transmission but Not Performance During Uphill and Downhill Mountain Biking over Rough Terrain.","authors":"Enrique Moreno-Manas, Salvador Llana-Belloch, Gonzalo Monfort-Torres, Xavier García-Massó","doi":"10.3390/mps9040118","DOIUrl":"10.3390/mps9040118","url":null,"abstract":"<p><p>Mountain biking over rough terrain exposes riders to vibrations that may affect comfort, health, bicycle control, and performance. This study analyzed the effect of wheel diameter on vibration transmission and performance during a short uphill and downhill test over rocky terrain. Forty-nine highly trained male mountain bikers completed repeated trials using two equivalent hardtail mountain bikes with 26- and 29-inch wheels. Vibrations were recorded with eight triaxial accelerometers placed on the bicycle and rider, and performance was assessed using an electronic photocell timing system. Both wheel sizes showed a similar vibration pattern, with lower root mean square (RMS) acceleration values at the helmet and coccyx and higher values at the wrists and ankles. However, wheel diameter significantly influenced vibration transmission. During the uphill test, the 26-inch bicycle produced higher accelerations at the coccyx and rear hub, whereas during the downhill test, the 29-inch bicycle transmitted greater vibrations to the rider's wrists and ankles. No significant differences were observed between wheel sizes in the time required to complete either the uphill or downhill tests. These findings suggest that, in short and highly irregular uphill and downhill sections, 29-inch wheels do not necessarily improve performance over 26-inch wheels, although they modify the distribution of vibrations transmitted to the rider.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13515466/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148830483","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Ultrasound-Enhanced Chemical Tissue Clearing of Large Specimens for 3D Microscopy: A Rapid and Accessible Method Using Standard Laboratory Equipment.","authors":"Klaus Becker, Seyed Meraaj Foroughipour, Massih Foroughipour, Karoline Maria Schwendt, Stefan H Geyer, James Oakes-Klein, Christoph Fuchssteiner, Wolfgang J Weninger, Eugenijus Kaniusas, Saiedeh Saghafi","doi":"10.3390/mps9040119","DOIUrl":"10.3390/mps9040119","url":null,"abstract":"<p><p>While the recently developed tissue clearing protocols pathoDISCO and activeDISCO significantly accelerate the clearing of large tissue specimens through active chemical dehydration using 2,2-dimethoxypropane, the final step of refractive index (RI) matching with viscous organic solvents as dibenzyl ether (DBE) remains restricted by slow passive diffusion. To overcome this bottleneck, we applied 40 kHz ultrasound using a standard, cost-effective laboratory bath to significantly enhance the diffusion kinetics of the clearing medium into large specimens. Our investigation on multi-centimeter-sized porcine muscle and human earlobe samples demonstrates that 40 kHz acoustic oscillations generated by a standard ultrasound cleaning device not only accelerate the clearing process but also yield superior and stable long-term optical transparency. We also tested 1 MHz high-frequency ultrasound but it offered no kinetic advantages and tended to induce tissue micro-fractures, an artifact we have not observed at 40 kHz at comparable energy levels. We therefore propose that standard 40 kHz ultrasound baths, ubiquitous in laboratories for cleaning purposes, represent an ideal and accessible tool for optimizing solvent-based tissue clearing.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13517022/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148830540","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}