Tissue Fixation Research Articles

The Measurement of Bovine Pericardium Density and Its Implications on Leaflet Stress Distribution in Bioprosthetic Heart Valves.

Bioprosthetic Heart Valves (BHVs) are widely used in clinical practice, showing promising outcomes. Computational modeling offers a valuable tool for quantitatively characterizing BHVs. To ensure the accuracy of computational models, it is crucial to consider precise leaflet properties, including mechanical properties and density. Bovine pericardium (BP) serves as a common material for BHV leaflets. Previous computational studies often assume BP density to approximate that of water or blood. Given that BP leaflets undergo various treatments, such as tissue fixation and anti-calcification, this study aims to measure the density of BP used in BHVs and assess its impact on leaflet stress distribution. Eight square BP samples were laser cut from Edwards BP patches and their density was determined. Specimen weight was measured using an A&D Analytical Balance, while volume was assessed through high-resolution imaging. Additionally, finite element models resembling a BHV, like the Carpentier-Edwards PERIMOUNT Magna, were constructed in ABAQUS. The average density of the BP samples was found to be 1,410kg/m3. During the acceleration phase of a cardiac cycle, the maximum stress reached 1.89MPa for a density of 1,410kg/m3 and 2.47MPa for a density of 1,000kg/m3 (a 30.7% difference). In the deceleration phase, the maximum stress reached 713kPa and 669kPa, respectively. Leaflet stress distribution and motion in BHVs are influenced by density variations. Establishing an accurate density value for BHV leaflets is imperative for enhancing the computational models, which can ultimately contribute to improved BHV design and outcomes.

Live Mapping of the Brain Extracellular Matrix and Remodeling in Neurological Disorders.

Live imaging of the brain extracellular matrix (ECM) provides vital insights into changes that occur in neurological disorders. Current techniques such as second or third-harmonic generation offer limited contrast for live imaging of the brain ECM. Here, a new method, pan-ECM via chemical labeling of extracellular proteins, is introduced for live brain ECM imaging. pan-ECM labels all major ECM components in live tissue including the interstitial matrix, basement membrane, and perineuronal nets. pan-ECM enables in vivo observation of the ECM heterogeneity between the glioma core and margin, as well as the assessment of ECM deterioration under stroke condition, without ECM shrinkage from tissue fixation. These findings indicate that the pan-ECM approach is a novel way to image the entire brain ECM in live brain tissue with optical resolution. pan-ECM has the potential to advance the understanding of ECM in brain function and neurological diseases.

Alternative Tissue Fixation Protocols Dramatically Reduce the Impact of DNA Artifacts, Unraveling the Interpretation of Clinical Comprehensive Genomic Profiling

Formalin-fixed paraffin-embedded (FFPE) samples represent the cornerstone of tissue-based analysis in precision medicine. Targeted next-generation sequencing panels are routinely used to analyze a limited number of genes to guide treatment decision-making for advanced-stage patients. The number and complexity of genetic alterations to be investigated are rapidly growing; in several instances, a comprehensive genomic profiling analysis is needed. The poor quality of genetic material extracted from FFPE samples may impact the feasibility/reliability of sequencing data. We sampled 9 colorectal cancers to allow 4 parallel fixations: (1) neutral buffered formalin (NBF), (2) acid-deprived formalin fixation (ADF), (3) precooled ADF (coldADF), and (4) glyoxal acid free (GAF). DNA extraction, fragmentation analysis, and sequencing by 2 large next-generation sequencing panels (OCAv3 and TSO500) followed. We comprehensively analyzed library and sequencing quality controls and the quality of sequencing results. Libraries from coldADF samples showed significantly longer reads than the others with both panels. ADF-derived and coldADF-derived libraries showed the lowest level of noise and the highest levels of uniformity with the OCAv3 panel, followed by GAF and NBF samples. The data uniformity was confirmed by the TSO500 results, which also highlighted the best performance in terms of the total region sequenced for the ADF and coldADF samples. NBF samples had a significantly smaller region sequenced and displayed a significantly lower number of evaluable microsatellite loci and a significant increase in single-nucleotide variations compared with other protocols. Mutational signature 1 (aging and FFPE artifact related) showed the highest (37%) and lowest (17%) values in the NBF and coldADF samples, respectively. Most of the identified genetic alterations were shared by all samples in each lesion. Five genes showed a different mutational status across samples and/or panels: 4 discordant results involved NBF samples. In conclusion, acid-deprived fixatives (GAF and ADF) guarantee the highest DNA preservation/sequencing performance, thus allowing more complex molecular profiling of tissue samples.

Beginners guide to sample preparation techniques for transmission electron microscopy

Background purpose: The revolution in microscopy came in 1930 with the invention of electron microscope. Since then, we can study specimens on ultrastructural and even atomic level. Besides transmission electron microscopy (TEM), for which specimen preparation techniques will be described in this article, there are also other types of electron microscopes that are not discussed in this review. Materials and methods: Here, we have described basic procedures for TEM sample preparation, which include tissue sample preparation, chemical fixation of tissue with fixatives, cryo-fixation performed by quick freezing, dehydration with ethanol, infiltration with transitional solvents, resin embedding and polymerization, processing of embedded specimens, sectioning of samples with ultramicrotome, positive and negative contrasting of samples, immunolabeling, and imaging. Conclusion: Such collection of methods can be useful for novices in transmission electron microscopy.

Shadow imaging for panoptical visualization of brain tissue in vivo

Progress in neuroscience research hinges on technical advances in visualizing living brain tissue with high fidelity and facility. Current neuroanatomical imaging approaches either require tissue fixation (electron microscopy), do not have cellular resolution (magnetic resonance imaging) or only give a fragmented view (fluorescence microscopy). Here, we show how regular light microscopy together with fluorescence labeling of the interstitial fluid in the extracellular space provide comprehensive optical access in real-time to the anatomical complexity and dynamics of living brain tissue at submicron scale. Using several common fluorescence microscopy modalities (confocal, light-sheet and 2-photon microscopy) in mouse organotypic and acute brain slices and the intact mouse brain in vivo, we demonstrate the value of this straightforward ‘shadow imaging’ approach by revealing neurons, microglia, tumor cells and blood capillaries together with their complete anatomical tissue contexts. In addition, we provide quantifications of perivascular spaces and the volume fraction of the extracellular space of brain tissue in vivo.

Combinatorial fluorescent labeling of live anaerobic bacteria via the incorporation of azide-modified sugars into newly synthesized macromolecules.

The human gut microbiome modulates physiological functions and pathologies; however, a mechanistic understanding of microbe-host and microbe-microbe interactions remains elusive owing to a lack of suitable approaches to monitor obligate anaerobic bacterial populations. Common genetically encoded fluorescent protein reporters, derived from the green fluorescent protein, require an oxidation step for fluorescent light emission and therefore are not suitable for use in anaerobic microbes residing in the intestine. Fluorescence in situ hybridization is a useful alternative to visualize bacterial communities in their natural niche; however, it requires tissue fixation. We therefore developed an approach for the real-time detection and monitoring of live communities of anaerobic gut commensals in their natural environment. We leverage the bacterial cells' reliance on sugars for macromolecule synthesis in combinatorial click chemistry labeling, where the addition of azide-modified sugars to the culturing media enables the fluorescence labeling of newly synthesized molecules via the addition of combinations of exogenous fluorophores conjugated to cyclooctynes. This process is suitable for labeling communities of live anaerobic gut bacteria with combinations of fluorophores that do not require oxygen to mature and fluoresce, and that can be detected over time in their natural environments. The labeling procedure requires 4-9 d, depending on the varying growth rates of different bacterial strains, and an additional 1-2 d for the detection and monitoring steps. The protocol can be completed by users with basic expertise in bacterial culturing.

Microvascular Endothelial Glycocalyx Surface Layer Visualization and Quantification.

As a primary interface between the blood and underlying vascular wall, the endothelial glycocalyx layer is common to all blood vessels and covers the luminal surface of all endothelial cells. The endothelial glycocalyx has important roles as a regulator of microvascular endothelial functions such as mechanotransduction, leukocyte adhesion, and microvascular permeability. Disruption of the molecular structure of the endothelial glycocalyx disturbs physiological, and hemodynamic processes associated with the microvascular wall leads to microvascular hyperpermeability. Studying the glycocalyx is challenging because cultured cells present aberrant glycocalyx structure and tissue fixation techniques lead to the degradation and loss of this fine and delicate layer. Therefore, studying the glycocalyx requires in vivo imaging of the microcirculation. Here we describe two techniques for direct imaging and assessment of the glycocalyx surface layer integrity using intravital microscopy (IVM), a method widely used in the study of the dynamic changes that occur in the microcirculation during inflammation or injury.

Optimization of Tissue Handling and Processing in the Era of Precision Medicine: A Practical Recommendation from a Multidisciplinary Panel of Indian Experts

AbstractMolecular analysis of biospecimens is the key to diagnostic and therapeutic decisions in clinical practice. However, there is a lack of consolidated guidelines for biospecimen collection, tissue handling, and storage in India. Therefore, this study aims to generate expert recommendations for the optimization of tissue handling and processing practices in India in the era of precision medicine. This study aimed to evaluate the clinical gaps related to tissue handling for molecular analysis and develop expert recommendations to mitigate preanalytical issues associated with biospecimen processing. These expert recommendations will help in increasing the diagnostic yield and accuracy of biomarker testing in clinical practice. A virtual advisory board meeting was convened with 19 experts, including pathologists, molecular biologists, medical oncologists, surgical oncologists, interventional radiologists, and a senior histology technician from 10 hospitals in India, along with an accreditation officer for testing and calibration of laboratory procedures. The scientific coordinators developed specific questions to address the salient issues associated with the preanalytic phase of tissue specimen preparation. The experts discussed each question until a complete set of recommendations was obtained. The expert panel provided recommendations for tissue collection, processing, fixation, and block preparation to ensure high-quality biospecimens. As per the expert panel recommendations, tissue sampling can be performed from any easily accessible site, regardless of the primary or metastatic locations. In addition, the cold ischemia time should be <1 hour, 10% neutral-buffered formalin should be used as the fixative, isopropyl alcohol should be used as the dehydrating agent, the volume of tissue to fixative ratio should be 1:10, and all the paraffin blocks should be archived in dry, pest-free conditions at room temperature. The experts suggested that the formalin used for fixation should be freshly prepared and its pH should be checked daily; moreover, the pH and date of formalin preparation should be mentioned on the containers. The experts highlighted the need to educate multidisciplinary teams on the optimization of tissue handling practices and emphasized that a pathologist should always check the tissue for adequate quality and quantity for biomarker testing. The existing routine clinical procedures for collecting and handling biospecimens adversely affect their quality. The expert recommendations for preanalytical quality control would ensure high-quality biospecimens for molecular analysis and precision medicine.

Medial Meniscal Allograft Transplantation: The Bone Plug Technique

Background: The medial and lateral menisci act as shock absorbers for the knee joint by converting and redistributing axial load into circumferential hoop stresses. Disruptions of these hoop stresses occur in the setting of meniscal deficiency and lead to long-term degenerative changes. Therefore, maintaining the distinctive composition and organization of the menisci is essential. In selective cases of meniscal deficiency, meniscus allograft transplantation can be a valuable treatment option. Indications: Meniscus transplantation should be considered in patients with symptomatic meniscal deficiency, without the presence of advanced degenerative pathologies, who have failed all conservative treatments. Technique description: We can divide the surgery into 4 steps: (1) graft preparation, (2) arthroscopic joint preparation, (3) allograft attachment preparation, and (4) graft fixation. Results: Meniscus allograft transplantation yields good to excellent results in up to 85% of cases. Improvement of pain and knee function occurs in approximately 70% of the patients at 10 years. The associated complications are mainly joint stiffness, early osteoarthritis, and incomplete healing accompanied by graft failure. Graft failure is the most feared complication, yet shows good results over the midterm. Conclusion: The bone plug technique we have shown here is a hybrid approach combining soft tissue and bone fixation techniques. It provides synergistic advantages with good osseous integration and is minimally invasive through arthroscopy without true arthrotomy. In our experience, this approach elegantly eases the complexity of this demanding surgery while yielding excellent results for patients. Patient Consent Disclosure Statement: The author(s) attests that consent has been obtained from any patient(s) appearing in this publication. If the individual may be identifiable, the author(s) has included a statement of release or other written form of approval from the patient(s) with this submission for publication.

Brain Stiffness Follows Cuprizone-Induced Variations in Local Myelin Content

Brain maturation and neurological diseases are intricately linked to microstructural changes that inherently affect the brain’s mechanical behavior. Animal models are frequently used to explore relative brain stiffness changes as a function of underlying microstructure. Here, we are using the cuprizone mouse model to study indentation-derived stiffness changes resulting from acute and chronic demyelination during a 15-week observation period. We focus on the corpus callosum, cingulum, and cortex which undergo different degrees of de- and remyelination and, therefore, result in region-specific stiffness changes. Mean stiffness of the corpus callosum starts at 1.1 ± 0.3 kPa in untreated mice, then cuprizone treatment causes stiffness to drop to 0.6 ± 0.1 kPa by week 3, temporarily increase to 0.9 ± 0.3 kPa by week 6, and ultimately stabilize around 0.7 ± 0.1 kPa by week 9 for the rest of the observation period. The cingulum starts at 3.2 ± 0.9 kPa, then drops to 1.6 ± 0.4 kPa by week 3, and then gradually stabilizes around 1.4 ± 0.3 kPa by week 9. Cortical stiffness exhibits less stiffness variations overall; it starts at 4.2 ± 1.3 kPa, drops to 2.4 ± 0.6 kPa by week 3, and stabilizes around 2.7 ± 0.9 kPa by week 6. We also assess the impact of tissue fixation on indentation-based mechanical tissue characterization. On the one hand, fixation drastically increases untreated mean tissue stiffness by a factor of 3.3 for the corpus callosum, 2.9 for the cingulum, and 3.6 for the cortex; on the other hand, fixation influences interregional stiffness ratios during demyelination, thus suggesting that fixation affects individual brain tissues differently. Lastly, we determine the spatial correlation between stiffness measurements and myelin density and observe a region-specific proportionality between myelin content and tissue stiffness. Statement of significanceDespite extensive work, the relationship between microstructure and mechanical behavior in the brain remains mostly unknown. Additionally, the existing variation of measurement results reported in literature requires in depth investigation of the impact of individual cell and protein populations on tissue stiffness and interregional stiffness ratios. Here, we used microindentation measurements to show that brain stiffness changes with myelin density in the cuprizone-based demyelination mouse model. Moreover, we explored the impact of tissue fixation prior to mechanical characterization because of conflicting results reported in literature. We observe that fixation has a distinctly different impact on our three regions of interest, thus causing region-specific tissue stiffening and, more importantly, changing interregional stiffness ratios.

An Ex Vivo Porcine Model for Hydrodynamic Testing of Experimental Aortic Valve Procedures and Novel Medical Devices.

The options for testing new cardiac procedures and investigative medical devices prior to use in an animal model are limited. In this study, we present a method for mounting a porcine aortic valve in a pulse duplicator to evaluate its hydrodynamic properties. These properties can then be evaluated before and after the procedure under investigation is performed and/or the investigative medical device is applied. Securing the inflow segment presents some difficulty owing to the lack of circumferential myocardium in the left ventricular outflow tract. This method addresses that issue by securing the inflow segment using the anterior leaflet of the mitral valve and then suturing the left ventricular free wall around the inflow fixture. The outflow segment is secured simply by inserting the fixture into an incision in the superior aspect of the aortic arch. We found that specimens had significantly different hydrodynamic properties before and after tissue fixation. This finding induced us to use fresh specimens in our testing and should be considered when using this method. In our work, we used this method to test novel intracardiac patch materials for use in the valvular position by performing an aortic valve neocuspidization procedure (Ozaki procedure) on the mounted porcine aortic valves. These valves were tested before and after the procedure to assess the change in hydrodynamic properties in comparison to the native valve. Herein, we report a platform for hydrodynamic testing of experimental aortic valve procedures that enables comparison with the native valve and between different devices and techniques used for the procedure under investigation.

Effect of uncertain clinical conditions on the early healing and stability of distal radius fractures

Background and objectivesThe healing outcomes of distal radius fracture (DRF) treated with the volar locking plate (VLP) depend on surgical strategies and postoperative rehabilitation. However, the accurate prediction of healing outcomes is challenging due to a range of certainties related to the clinical conditions of DRF patients, including fracture geometry, fixation configuration, and physiological loading. The purpose of this study is to investigate the influence of uncertainty and variability in fracture/fixation parameters on the mechano-biology and biomechanical stability of DRF, using a probabilistic numerical approach based on the results from a series of experimental tests performed in this study. MethodsSix composite radius sawboneses fitted with titanium VLP (VLP 2.0, Austofix) were loaded to failure at a rate of 2 N/s. The testing results of the elastic and plastic behaviour of the VLP were used as inputs for a probabilistic-based computational model of DRF, which simulated mechano-regulated tissue differentiation and fixation elastic capacity at the fracture site. Finally, the probability of success in early indirect healing and fracture stabilisation was predicted. ResultsThe titanium VLP is a strong and ductile fixation whose flexibility and elastic capacity are governed by flexion working length and bone-to-plate distance, respectively. A fixation with optimised designs and configurations is critical to mechanically stabilising the early fracture site. Importantly, the uncertainty and variability in fracture/fixation parameters could compromise early DRF healing. The physiological loading uncertainty is the most adverse factor, followed by the negative impact of uncertainty in fracture geometry. ConclusionsThe VRP 2.0 fixation made of grade II titanium is a desirable fixation that is strong enough to resist irreparable deformation during early recovery and is also ductile to deform plastically without implant failure at late rehabilitation.

Rapid in-solution preparation of somatic and meiotic plant cell nuclei for high-quality 3D immunoFISH and immunoFISH-GISH

BackgroundThough multicolour labelling methods allow the routine detection of a wide range of fluorescent (immuno)probe types in molecular cytogenetics, combined applications for the simultaneous in situ detection of proteins and nucleic acids are still sporadic in plant cell biology. A major bottleneck has been the availability of high-quality plant nuclei with a balance between preservation of 3D ultrastructure and maintaining immunoreactivity.The aim of this study was to develop a quick and reliable procedure to prepare plant nuclei suitable for various combinations of immunolabelling and fluorescence in situ hybridisation methods (immunoFISH-GISH).ResultsThe mechanical removal of the cell wall and cytoplasm, instead of enzymatic degradation, resulted in a gentle, yet effective, cell permeabilisation. Rather than manually releasing the nuclei from the fixed tissues, the procedure involves in-solution cell handling throughout the fixation and the preparation steps as ended with pipetting the pure nuclei suspension onto microscope slides. The optimisation of several critical steps is described in detail. Finally, the procedure is shown to be compatible with immunolabelling, FISH and GISH as well as their simultaneous combinations.ConclusionA simple plant cell nuclei preparation procedure was developed for combined immunolabelling-in situ hybridisation methods. The main and critical elements of the procedure are: a short period of fixation, incorporation of detergents to facilitate the fixation of tissues and the penetration of probes, tissue grinding to eliminate unwanted cell components, and an optimal buffer to handle nuclei. The procedure is time efficient and is easily transferable without prior expertise.

A Radiostereometric Analysis of Tendon Migration After Arthroscopic and Mini-Open Biceps Tenodesis: Interference Screw Versus Single Suture Anchor Fixation.

Studies suggest that similar clinical results are achieved via arthroscopic and open biceps tenodesis (BT) techniques. To quantify the postoperative migration of the BT construct between arthroscopic suprapectoral BT (ASPBT) and open subpectoral BT (OSPBT) techniques via interference screw (IS) or single-suture suture anchor (SSSA) fixation using radiostereometric analysis. Cohort study; Level of evidence, 2. Distal migration of the biceps tendon after OSPBT with a polyetheretherketone IS, OSPBT with 1 SSSA, ASPBT with polyetheretherketone IS, and ASPBT with 2 SSSAs was measured prospectively. Patients with symptomatic biceps tendinopathy and preoperative patient-reported outcome measures (PROMs) including Constant-Murley subjective, Single Assessment Numeric Evaluation, or Patient-Reported Outcomes Measurement Information System-Upper Extremity scores were included. A tantalum bead was sutured on the proximal end of the long head of the biceps tendon before fixation of tendon tissue. Anteroposterior radiographs were performed immediately postoperatively, at 1 week, and at 3 months. Bead migration was measured, and preoperative PROMs were compared with those at latest follow-up. Of 115 patients, 94 (82%) were available for final follow-up. IS fixation yielded the least tendon migration with no difference between the open and arthroscopic approaches (4.31 vs 5.04 mm; P = .70). Fixation with 1 suture anchor demonstrated significantly greater migration than that achieved with an IS at both 1 week (6.47 vs 0.1 mm, 6.47 vs 1.75 mm, P < .001;) and 3 months (14.76 vs 4.31 mm, 14.76 vs 5.04 mm, P < .001) postoperatively. Two-suture anchor fixation yielded significantly greater migration than IS fixation at 1 week (7.02 vs 0.1 mm, P < .001; 7.02 vs 1.75 mm, P = .003) but not 3 months postoperatively (8.06 vs 4.31 mm, P = .10; 8.06 vs 5.04 mm, P = .07). Four patients with suture anchor fixation (3 patients in the OSPBT 1 SSSA group, 9.4%, and 1 patient in the ASPBT 2 SSSAs group, 3.8%) developed a Popeye deformity, whereas no Popeye deformities occurred in the IS groups. Mean 3-month bead migration in patients with and without a Popeye deformity was 60.8 and 11.2 mm, respectively (P < .0001). PROMs did not differ among groups at final follow-up. Interference screw fixation yielded the least tendon migration whether achieved arthroscopically or open. The available data indicated that fixation with 1 SSSA but not 2 SSSAs resulted in significantly greater migration than that achieved with an IS. Despite variations in tendon migration, PROMs were similar among all groups. When SSSAs are used, tendon migration may be minimized by using ≥2 anchors.

Formalin Versus Bouin Solution for Rat Testicular Tissue Fixation: A Histochemical and Immunohistochemical Evaluation

Background: An accurate histopathological assessment and reporting of testicular biopsies require an appropriate tissue fixative. We assessed the histological, histochemical, and immunohistochemical quality of testicular biopsies, comparing 10% formalin versus Bouin solution as tissue fixatives. Methods: This experimental study utilized 20 adult male albino rats equally divided into five cages for 30 days. By the end of the experiment, all animals were anesthetized, and both testes were removed and weighted; one testicle was fixed in 10% formalin and the other testicle in Bouin solution, offering 40 specimens and then subjected to histological, morphometric, histochemical, and immunohistochemical assessments. Results: Formalin revealed high-quality cytological details and better nuclear chromatin detail (P=0.03). At the architectural level, the Bouin solution showed better quality details with less cytoplasmic shrinkage of seminiferous tubule germ cells (P=0.001). Bouin’s fixed tissues were more suitable for staining by trichrome methods but unsuitable when subsequent immunohistochemistry was requested. The diagnostic concordance between the Bouin solution versus formalin-fixed biopsies was 91.7%. Conclusion: This study supports that the morphology of testicular tissue fixed with Bouin solution was nearly comparable to those fixed with 10% neutral buffered formalin. However, the Bouin solution cannot substitute formalin when subsequent immunohistochemistry is considered.

The Efficacy and Safety of Single-Incision Mini-Slings for Stress Urinary Incontinence: A Network Meta-Analysis.

To evaluate the efficacy and safety of single-incision mini-sling for stress urinary incontinence based on network Meta-analysis. We searched PubMed, Embase, and Cochrane libraries from August 2008 to August 2019. Randomized controlled trials comparing two or more indicators of Miniarc (Single Incision Mini-slings), Ajust (Adjustable Single-Incision Sling), C-NDL (Contasure-Needleless), TFS (Tissue Fixation System), Ophria (Transobturator Vaginal Tap), TVT-O (Transobturator Vaginal Tape), and TOT (Trans-obturatortape) in treating female stress urinary incontinence were collected. Totally, 3,428 patients from 21 studies were included. Ajust had the highest subjective cure rate (Rank=0.52), while Ophira had the worst (Rank=0.67). TFS had the highest objective cure rate, and the worst was found in Ophira. TFS required the shortest operating time (Rank=0.40), while TVT-O required the longest operating time (Rank=0.47). Miniarc had the least bleeding (Rank=0.47), while TVT-O had the most bleeding (Rank=0.37). C-NDL had the shortest postoperative hospital stay (Rank=0.77), while Ajust had the longest postoperative hospital stay (Rank=0.36). For postoperative complications, TFS performed best in groin pain (Rank=0.84), urinary retention (Rank=0.78), and repeat surgery (Rank=0.45). TVT-O performed worst in groin pain (Rank=0.36), and urinary retention (Rank=0.58). Miniarc had the highest repeat surgery rate (Rank=0.35). Ajust had the lowest probability of tap erosion (Rank=0.30), while Ophira had the highest tap erosion level (Rank=0.45). Miniarc showed the greatest advantage in urinary tract infections (Rank=0.84) and de novo urgency (Rank=0.60), while C-NDL had the highest incidence of urethral infections (Rank=0.51). Ophira performed worst in de novo urgency (Rank=0.60). C-NDL performed the best in sexual intercourse pain (Rank=0.79) while Ajust was the worst (Rank=0.49). In view of comprehensive efficacy and safety, we recommend that TFS or Ajust should be selected first for single-incision sling and the application of Ophria should be minimized.

Effectiveness of neutral honey as a tissue fixative in histopathology

Background: In routine histopathology, 10% neutral buffered formalin (NBF) is the choice fixative. However, formalin is a human carcinogen, so there is a necessity for a safer alternative. To the best of our knowledge, neutral honey, where the pH is around seven, not natural or artificial honey, has not been tested to fix histological samples. This study determined the effectiveness of neutral buffered honey and other types of fixatives to fix histological tissues. Methods: The study was conducted between July 2019 and August 2020 at Sultan Qaboos University, Oman. Sections from three rat livers, kidneys, and stomach tissues were fixed with 10% NBF, neutral buffered Sumer honey (dwarf honey bee), neutral buffered date honey, formalin, Sumer honey, date honey, alcoholic formalin, alcoholic Sumer honey, and alcoholic date honey for 24 hours. Samples were stained with hematoxylin and eosin (H&amp;E), special stains, and vimentin methods. Three expert biomedical scientists then evaluated the fixed and stained samples for the quality of all sections. The fixation ability of the different honey solutions was then compared to 10% NBF and the utility was determined using nuclear and cytoplasmic criteria, specificity, and intensity. Results: H&amp;E showed adequate nuclear staining in all groups compared to 10% NBF. The specificity and intensity of all groups for the Periodic acid–Schiff method were identical to 10% NBF except for Sumer honey and alcoholic date honey. Vimentin showed comparable findings with 10% NBF as there were no significant differences. Conclusions: The findings of this study encourage the use of honey, including neutral, as a possible safe substitute fixative for formalin, however, further experiments on larger specimens should be conducted.

Effect of tissue fixation on the optical properties of structural components assessed by non-linear microscopy imaging.

Fixation methods such as formalin are commonly used for the preservation of tissue with the aim of keeping their structure as close as possible to the native condition. However, fixatives chemically interact with tissue molecules, such as collagen in the extracellular matrix (ECM) or myosin, and may thus modify their structure. Taking advantage of the second- and third-harmonic generation (SHG and THG) emission capabilities of such components, we used nonlinear two-photon microscopy (NL2PM) to evaluate the effect that preservation methods, such as chemical fixatives, have on the nonlinear capabilities of protein components within mouse tissues. Our results show that depending on the preservation technique used, the nonlinear capabilities of collagen, lipid droplets and myosin microarchitecture are strongly affected. Parameters of collagen fibers, such as density and branch points, especially in collagen-sparse regions, e.g., in kidneys, were found to be altered upon formalin fixation. Moreover, cryo-freezing drastically reduced SHG signals from myosin. Our findings provide valuable information to select the best tissue fixation method for visualization and quantification of structural proteins, such as collagen and myosin by advanced NL2PM imaging techniques. This may advance the interpretation of the role these proteins play in disease.

A comparative analysis of two antigen retrieval techniques: Microwave oven and pressure cooker for immunoexpression of estrogen and progesterone receptors in breast cancer tissue

Objectives: Antigen retrieval (AR) is an important step in Immunohistochemistry (IHC) which is used to unmask the antigenic sites and facilitate antigen-antibody binding. Adequate fixation of tissue is necessary to achieve consistent demonstration of tissue antigens that can be masked by the chemical process involved in formalin fixation and tissue processing. Out of the various methods of AR, heat-induced epitope retrieval (HIER) methods have greatly improved the quality and reproducibility of IHC. In this study, a comparison of the two most commonly used HIER methods-pressure cooker and microwave oven was done on thirty cases of breast carcinoma. Materials and Methods: Appropriate tumor sections were taken and subjected to manual IHC testing for estrogen receptor (ER) and progesterone receptor (PR) receptors in each case. The results were divided into technique and microscopy-based. The parameters assessed on microscopy were uniformity of nuclear staining, quality of nuclear staining, internal control staining, presence of background staining, and Allred score. The sensitivity and specificity and positive and negative predictive values for each method were calculated. Results: The parameters assessed on microscopy were comparable for both methods. Using a microwave oven, the sensitivity and specificity for ER and PR were 94% and 100%, respectively. Using a pressure cooker, the sensitivity, and specificity for ER were 94% and 100%, respectively, and for PR were 88% and 100%, respectively. On technical aspects, the pressure cooker method offers the advantage of being more convenient due to the possibility of simultaneous handling of more slides and being more time efficient. Conclusion: Both the AR methods had comparable results on microscopy. However, the pressure cooker has the benefit of being both time and money efficient from a technical standpoint.

Fixation and Laser Capture Microdissection of Plant Tissue for RNA Extraction and RNASeq Library Preparation.

To study the transcriptome of individual plant cells at specific points in time, we developed protocols for fixation, embedding, and sectioning of plant tissue followed by laser capture microdissection (LCM) and processing for RNA recovery. LCM allows the isolation of individual cell types from heterogeneous tissue sections and is particularly suited to plant processing because it does not require the breakdown of cell walls. This approach allows accurate separation of a small volume of cells that can be used to study gene expression profiles in different tissues or cell layers. The technique requires neither separation of cells by enzymatic digestion of any kind nor cell-specific reporter genes, and it allows storage of fixed and embedded tissue for months before capture. The methods for fixation, embedding, sectioning, and capturing of plant cells that we describe yield high-quality RNA suitable for making libraries for RNASeq. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Tissue Preparation for Laser Capture Microdissection Basic Protocol 2: Tissue Sectioning Basic Protocol 3: Laser Capture Microdissection of Embedded Tissue Basic Protocol 4: RNA Extraction from Laser Capture Microdissection Samples.