Coverslip Removal Research Articles

A machine learning approach toward automating spatial identification of LAG3+/CD3+ cells in ulcerative colitis

Over the past decade, automation of digital image analysis has become commonplace in both research and clinical settings. Spurred by recent advances in artificial intelligence and machine learning (AI/ML), tissue sub-compartments and cellular phenotypes within those compartments can be identified with higher throughput and accuracy than ever before. Recently, immune checkpoints have emerged as potential targets for auto-immune diseases. As such, spatial identification of these proteins along with immune cell markers (e.g., CD3+/LAG3+ T-cells) is a crucial step in understanding the potential and/or efficacy of such treatments. Here, we describe a semi-automated imaging and analysis pipeline that identifies CD3+/LAG3+ cells in colorectal tissue sub-compartments. While chromogenic staining has been a clinical mainstay and the resulting brightfield images have been utilized in AI/ML approaches in the past, there are associated drawbacks in phenotyping algorithms that can be overcome by fluorescence imaging. To address these tradeoffs, we developed an analysis pipeline combining the strengths of brightfield and fluorescence images. In this assay, immunofluorescence imaging was conducted to identify phenotypes followed by coverslip removal and hematoxylin and eosin staining of the same section to inform an AI/ML tissue segmentation algorithm. This assay proved to be robust in both tissue segmentation and phenotyping, was compatible with automated workflows, and revealed presence of LAG3+ T-cells in ulcerative colitis biopsies with spatial context preserved.

Role of dry ice in decoverslipping of microscopic slides: A new insight.

The process of decoverslipping is often required in a laboratory to review or examine an older slide which tends to fade over time, making it almost impossible to use it for research or study purposes. The sections then need to be re-stained which can only be done after removing the coverslip. The traditional method of decoverslipping using xylene is a time-consuming process. Various methods have been used in the past; however, none were found to be completely effective. Dry ice, the solid form of carbon dioxide, is an easily available, cheap cooling agent with a low freezing temperature (-78.5°C) which was evaluated for its efficacy in decoverslipping process, as an alternative to xylene. 64 faded haematoxylin and eosin (H&E)-stained histopathology slides were randomly selected and segregated, according to duration of year, into eight major groups. Each group was further divided into four subgroups according to the time that the slides were subjected for decoverslipping. The slides were placed on dry ice and the time was set. Once the coverslip was removed, the slides were placed in xylene to remove any residual mountant. The tissue sections were evaluated for physical disfigurement followed by re-staining with H&E to check for any change in tissue morphology. The mean time taken for removal of coverslip using dry ice was 35 seconds. This technique is easy, fast, and effective, with no tissue loss or compromise in staining quality, thereby preventing xylene toxicity and its effect on the environment.

DNA extraction and STR profiling from histological slides

Formalin-fixed paraffin-embedded (FFPE) tissue blocks are commonly used in the field of pathology and forensic pathology as a source of histological slides. For postmortem kinship analysis or identification, DNA can be extracted from blocks with specialized kits. However, when an STR profile should be generated from single microscope slides, the removal of the coverslip and the limited sample size poses unique challenges. We aimed to test the effectivity of agitated xylene incubation to dissolve the mounting material to facilitate the coverslip removal. DNA extraction tests were performed on 5- to 7-year-old histological slides. Xylol was used to dissolve the mounting medium to facilitate cover slide removal, one set of samples was shaken during incubation, and the other set was left still. It was found that shaking the sample while bathed in xylol decreased the incubation time from three days to two days. Agitation not just reduced the processing time but increased the quality of acquired STR profiles: on average 30% more alleles were detected from the shaken samples compared to the still bathed ones.

An assessment of decoverslipping methods on histology slides

ABSTRACTHistology laboratories are often required to decoverslip and restain older slides when the original staining on the section has faded or the mountant used for coverslipping has caused discoloration on the section. The current method of decoverslipping is time-consuming and three possible techniques for rapid coverslip removal have been compared for use in histopathology laboratories. Slides aged between 14 and 22 years were randomly selected to have their coverslip removed by liquid nitrogen, ultrasonic vibration, or freezing at −80°C. Both liquid nitrogen and −80°C cooling required a microtome blade to pry the coverslip from the slide following the rapid freezing. Liquid nitrogen and ultrasonic vibrations were both deemed unacceptable options for histological use due to cracking of the slide and loss of tissue. The cooling of slides in a −80°C freezer for 5–10 minutes had no tissue loss and the histological morphology remained intact. Therefore, freezing slides in a −80°C freezer is a suitable alternative from the commonly used xylene immersion removal.

Consecutive Immunohistochemical Staining on a Single Slide is Effective in Confirming Melanocytic Derivation.

Metastatic melanoma in sentinel lymph nodes is often elusive to detect with morphology alone. Per American Joint Committee on Cancer staging guidelines, a single atypical melanocyte in lymph node qualifies as metastasis, whether identified by morphology or immunohistochemistry, but single cell staining must be convincing. We propose that the use of a second immunohistochemical run performed on a single slide will allow for more confident diagnosis of micrometastases. We designed a technical study to determine whether a second antibody application on previously stained slides can successfully detect the same population of cells. Melanocytic neoplasms were stained with SOX-10 using Ventana Benchmark Ultra stainers, coverslipped, and examined, followed by coverslip removal and application of MART-1 (Ventana A103). The order of antibody application and chromagen detection kit (AP-RED vs. DAB) was reversed to establish reliability and robustness of the protocol. All melanocytes marked with SOX-10 and MART-1, and produced a range of staining quality that varied based on order of stain application and chromagen kit were used. The optimal combination was red MART-1 applied first followed by brown SOX-10 applied second. Consecutive staining of melanocytes with SOX-10 and MART-1 may improve diagnostic confidence of melanocyte identification, particularly in detection of single cell, micrometastases in sentinel lymph nodes or in situations where dual immunohistochemical stains may be unavailable.

Medium-retaining Petri dish insert to grow and image cultured cells

BackgroundMicroscope chambers that accept glass coverslips with cultured cells are often used to monitor intracellular Ca2+ concentration ([Ca2+]i) during cell superfusion. Unfortunately, the experimental maneuvers associated with the coverslip installation in these chambers (medium removal and re-application) trigger unintended [Ca2+]i elevations. New methodTo prevent these [Ca2+]i elevations, a Petri dish insert has been constructed. The insert features a superfusion-optimized well to grow cell cultures. After this insert is removed from the Petri dish, the well retains the medium. This feature allows the inserts to be installed in microscope chambers while keeping the cells submerged at all times. ResultsThese inserts were used to test the impact of a transient medium removal from the well (an equivalent of a coverslip removal from the medium) on [Ca2+]i in primary murine cortical neurons and astrocytes, and in HEK-293 cells. In all of these models, the medium removal/re-application caused a micromolar [Ca2+]i spike. While in neurons this spike was caused by a Ca2+ influx, in astrocytes and HEK-293 cells, it was caused by a Ca2+ release from intracellular stores. After the spike, a subpopulation of neurons failed to restore low [Ca2+]i; in 24% of the astrocytes, the spike triggered [Ca2+]i oscillations. However, prior to the spike, [Ca2+]i was low and uniform in all these cells. Comparison with existing method(s)The new method avoids the artificially-induced [Ca2+]i elevations that take place during the handling of glass coverslips with cultured cells. ConclusionsThe new method allows monitoring [Ca2+]i without disturbing the basal [Ca2+]i levels.

Resin-embedded Thin-section Immunohistochemistry Coupled with Triple Cellular Counterstaining.

This protocol was developed to study protein localisation within the vascular bundles of developing tomato fruit however, it can be applied to any resin embedded plant tissue. The vascular bundle is comprised of many different cells that all have unique properties. The mature sieve elements are enucleated and contain sieve plates that comprise of callose. This method has utilised these properties of the sieve element by combining immunohistochemistry for cell wall invertase with counterstaining of aniline blue for callose, DAPI for nucleus and cell structure is shown with the final staining of the cell wall using calcofluor white. It must be noted that when following this protocol, it is vital for the sections to be flat and fixed to the slide with gelatine so cover slip removal does not move the sample section. This protocol will be applicable to all plant tissues and provides additional evidence of the protein localisation within the cell by conducting a counterstaining procedure.

Coverslip Removal from Stained Cytology Slides for Molecular Testing Using a Hot Plate Method

Coverslip Removal from Stained Cytology Slides for Molecular Testing Using a Hot Plate Method

Rapid removal of cytology slide coverslips for DNA and RNA isolation

Because of consistent demand for identification of molecular targets for diagnosis and treatment, ancillary tests are increasingly being developed for cytology specimens. One time-consuming step in this procedure is glass coverslip removal by xylene. Here we describe a method for rapid coverslip removal using liquid nitrogen. Direct smears were prepared from residual pleural fluid, Diff-Quik stained, then covered with a glass coverslip. Coverslips were then removed by either immersing the slides in xylene for 3 to 4 days, or by a razor blade after rapidly freezing the slides in liquid nitrogen. The DNA or RNA was extracted, and the quantities of DNA and RNA were evaluated. The amplifiability of the DNA and RNA was also evaluated by real-time polymerase chain reaction (PCR) and real time reverse transcription (RT)-PCR. The concentrations of DNA isolated using the two procedures are not significantly different (2.79 μg/mL with liquid nitrogen method versus 2.56 μg/mL with xylene method, n = 5). Similar results were observed for RNA concentration (13.28 μg/mL versus 12.22 μg/mL). The DNA extracted from the two procedures can be amplified without significant difference. Similar results were obtained from real time RT-PCR for RNA samples. By removing the coverslip from the cytology slides using the liquid nitrogen method, DNA and RNA can be extracted from the slides without affecting the quantity and quality of the nucleic acids, and is equivalent to the xylene coverslip removal method.

Inhibition of Mitochondrial Na+/Ca2+ Exchanger Suppresses Ischemia/Reperfusion-Induced Reentry in Monolayers of Cardiomyocytes

Introduction: Mitochondria are important organelles that regulate cytosolic [Ca2+] ([Ca2+]c) in cardiomyocytes and are thought to play a major role in Ca2+ overload and arrhythmogenesis during ischemia/reperfusion. Previously, we showed that inhibition of the mitochondrial Na+/Ca2+ exchanger (mNCE) using CGP-37157 increases mitochondrial [Ca2+] ([Ca2+]m) retention, and prevents delayed afterdepolarizations and triggered arrhythmias during Na+ overload induced by Na+/K+ ATPase inhibitor, ouabain, in vitro and in vivo. Here, we studied the dynamics of [Ca2+]m and [Ca2+]c during ischemia/reperfusion, and investigated the role of mNCE in ischemia/reperfusion-related reentry in monolayers of neonatal rat ventricular myocytes. Methods: Sarcolemmal electrical activity was recorded with a 464-photodiode array using voltage-sensitive dye, di-4-ANEPPS. Changes in [Ca2+]m and [Ca2+]c were observed using the ratiometric genetically-encoded mitochondrial and cytosolic [Ca2+] indicator, GEM-GECO, respectively. Ischemia was induced by covering the central region of the monolayer with a coverslip and reperfusion was prompted by coverslip removal. Results: As the coverslip was lowered on the monolayer, amplitude and conduction velocity of action potentials decreased over time until the ischemic region became inexcitable. During 1 hour of ischemia, [Ca2+]m and [Ca2+]c increased in a sigmoid fashion. Reperfusion of the monolayers with Tyrode's solution sharply lowered [Ca2+]m back to its initial values in less than 10 minutes. Wavelets and subsequently reentry occurred upon reperfusion in 6/7 monolayers. CGP-37157 (1µM), at reperfusion, prevented or shortened the duration of wavelets, reducing the occurrence of reentry to 2/5 monolayers. Viral overexpression of NCLX, the molecular candidate for mNCE, did not alter the incidence of reentry, compared to controls. Conclusion: The results reveal the kinetics of [Ca2+] during ischemia/reperfusion using a novel genetically encoded probe and demonstrate the cardioprotective effects of inhibition of mNCE, as CGP-37157 decreased dispersion of repolarization and suppressed reentry.

A Human Proteome Microarray Identifies that the Heterogeneous Nuclear Ribonucleoprotein K (hnRNP K) Recognizes the 5′ Terminal Sequence of the Hepatitis C Virus RNA

Stem-loop I (SL1) located in the 5' untranslated region of the hepatitis C virus (HCV) genome initiates binding to miR-122, a microRNA required for hepatitis HCV replication. However, proteins that bind SL1 remain elusive. In this study, we employed a human proteome microarray, comprised of ∼17,000 individually purified human proteins in full-length, and identified 313 proteins that recognize HCV SL1. Eighty-three of the identified proteins were annotated as liver-expressing proteins, and twelve of which were known to be associated with hepatitis virus. siRNA-induced silencing of eight out of 12 candidate genes led to at least 25% decrease in HCV replication efficiency. In particular, knockdown of heterogeneous nuclear ribonucleoprotein K (hnRNP K) reduced HCV replication in a concentration-dependent manner. Ultra-violet-crosslinking assay also showed that hnRNP K, which functions in pre-mRNA processing and transport, showed the strongest binding to the HCV SL1. We observed that hnRNP K, a nuclear protein, is relocated in the cytoplasm in HCV-expressing cells. Immunoprecipitation of the hnRNP K from Huh7.5 cells stably expressing HCV replicon resulted in the co-immunoprecipitation of SL1. This work identifies a cellular protein that could have an important role in the regulation of HCV RNA gene expression and metabolism.

Minimizing delays in DNA retrieval: The “freezer method” for glass coverslip removal. Letter to the editor regarding comparative study of epidermal growth factor receptor mutation analysis on cytology smears and surgical pathology specimens from primary and metastatic lung carcinomas

Minimizing delays in DNA retrieval: The “freezer method” for glass coverslip removal. Letter to the editor regarding comparative study of epidermal growth factor receptor mutation analysis on cytology smears and surgical pathology specimens from primary and metastatic lung carcinomas

Effects Of Induced Post-ischemic Phosphorylation On Action Potential Propagation In Mouse Neonatal Cardiomyocytes

Intracellular events triggered by protein phosphorylation and kinase translocation have been reported to help preventing larger cardiac tissue damage. Our objective was to determine if immediate changes in phosphorylation post-infarction, resembling clinical treatments could affect conduction velocity and further cardiac protection.Cardiomyocytes from 0-2 day old mice pups were grown on 64 Micro-Electrode Arrays (MEAs; MES, Germany). The MEAs recorded action potential (AP) propagation produced by neonatal cardiomyocytes isolated and culture for 2 days. Myocytes were subjected to ischemia by placing a 13mm glass round cover-slip over the preparation for 45 minutes and recordings were made during and after cover-slip removal. After the ischemic event, random groups were treated with 300nM TPA to induce phosphorylation for one hour. TPA was removed and both groups were incubated for 24 hours. Following incubation cells were subjected to another ischemic event. Recordings were done after the ischemic event and again thirty minutes later. Conduction velocity (CV) was averaged from all electrodes.Phosphorylation and kinase translocation has been known to protect cardiocytes during ischemic preconditioning. We now present conditions where phosphorylation can actually be detrimental if induced after an ischemic event. At the 24 hour point, the control group had recovered AP CV by 99±2% (mean±SE) compared to the TPA treated group which recovered to 56±8%. The TPA treated group then showed only an average of 14±7% decrease in AP CV and AP amplitude upon administering the second ischemic event whereas the control group AP CV decreased by an average of 55%±9. When induced early after an ischemic event, phosphorylation results protective to maintain CV but appears to become detrimental for cellular survival.

Karyotypes of two cytotypes of Paspalum quadrifarium Lam. (Poaceae): an alternative technique for small chromosomes in plants

Paspalum quadrifarium Lam. is a bunchgrass native to Uruguay, Argentina, and southern Brazil. Diploid, triploid, tetraploid and hexaploid cytotypes have been reported for this species of the Quadrifaria group of Paspalum. In this group, a high degree of cytogenetic homology between the genomes of several diploid species has been reported, based on meiotic pairing in interspecific hybrids; multivalent associations would thus be expected in polyploid hybrids. Karyotype analysis could provide useful information about the genomic architecture of polyploid plants; however, the fully condensed mitotic chromosomes of Paspalum do not provide enough morphological features for such an analysis. In this paper, we used mitotic prometaphase chromosomes treated with 70% acetic acid at 40 °C after cover slip removal. This process removes cytoplasm that remains from chromosome squashes and makes prometaphases available for karyological analysis. The karyotypes of a triploid (2n = 3x = 30) and a tetraploid (2n = 4x = 40) accession of Paspalum quadrifarium were studied using this technique, and evidence of segmental allopolyploidy was found in both cases. In both accessions, meiotic behavior was in accordance with that origin. This technique greatly improved the number and quality of analyzable metaphases and prometaphases on otherwise conventional slides and is recommended for plants with small chromosomes.

Improved Hematoxylin Staining for Algal Cytogenetics

The aceto-iron hematoxylin method of Wittmann for algal chromosomes was improved by replacing the chloral hydrate and slide heating steps with 5 N HC1 hydrolysis. Squashing of the unstained material under the coverslip followed by coverslip removal and air drying allowed rapid and more homogeneous staining. Additional procedures were developed to make the method easier and quicker. Good results were obtained for all algal species analyzed. Pit-connections of red algae were also clearly differentiated.

Analysis of DNA Levels During Gonomery in Early Cleavage Divisions of the Freshwater Copepod Mesocyclops Edax

Gonomery, the separate grouping of maternal and paternal chromosomes during the first few mitoses following fertilization (Fig. 1) and chromatin diminution, the fragmentation and elimination of whole chromosome regions during specific stages of early cleavage in all primordial cells of the soma, occur in several species of copepods, ascarid nematodes, ciliated protozoa, Japanese hagfish 9 and a few other invertebrates. Because of the marked loss of DNA from all presumptive somatic cells by selective chromatin diminution during the 4th cleavage division in M. edax, it was of interest to determine DNA levels in chromosomes during gonomery in anaphases preceding diminution (Fig. 1). It was also important to estimate the DNA content of sperm as an index of the relative contribution by the male genome at fertilization in this species.Mature males and egg-carrying females were fixed in 3:1 methanol/acetic acid for 3-5 mins, swollen in 45% acetic acid for 2-3 mins, squashed, frozen in liquid N2 for coverslip removal, thawed in 2 changes of absolute ETOH and air dried.

Recycling cells in FISH and immunocytology studies

▼We have previously published protocols for combined fluorescent in situ hybridization (FISH) and immunocytology of meiotic chromosomes (Ref.1). Here we highlight two aspects of our procedures that are beneficial to other similar systems: the use of commercially available glass multiwell slides and an effective antifading mounting agent. With multiwell slides, material can be re-stained with different probes and/or antibodies. The 15 µm plastic coating around each well provides uniform support, protects cells from damage during removal of coverslips and precludes the need for wax or nail polish barriers. No sealants are necessary, discoloured mounting medium can be refreshed and the three-dimensional structure of the cell may be preserved for confocal microscopy, if desired. One can determine the most effective combination of experimental parameters by testing concentration gradients, comparing different antibodies, or screening combinations of fluorochromes. Testing requires minimal material − 5−10 µ lp er well− and the slides are less likely to break than coverslips, the alternative approach to minimizing reagent volumes. Multiwell slides may be recycled by washing in detergent, sonicating in a bleach/detergent solution and rinsing in water. The slides should be washed with a commercial glass cleaner (Windex) and rinsed in water just before use to ensure adhesion of tissue. The older and more used the slide, the better the adhesion of new material. An example of sequential staining is shown (Fig. 1). In this meiotic prophase nucleus, where homologous chromosomes form proteinaceous cores (synaptonemal complexes) and undergo pairing and recombination, the centromeres are stained with anti-centromere serum from a CREST (calcinosis, Raynaud’s phenomenon, oesophageal dysmotility, telangiectasia syndrome) patient. Centromeres are visualized yellow−orange with Cy3-conjugated antihuman secondary antibody through a fluorescein isothiocyanate (FITC) filter. The pairing region of the ho

Detection of numerical chromosomal abnormalities by fluorescence in situ hybridization of interphase cell nuclei with chromosome-specific probes on archival cytologic samples.

Fluorescence in situ hybridization (FISH) is rapidly emerging as a tool for analyzing numerical and structural chromosomal abnormalities in both liquid and solid tumors. Most studies make use of fresh samples. To determine the feasibility of detecting numerical chromosomal abnormalities (NCA) by FISH using chromosome-specific probes 8, 12, 17, and X (Vysis, Inc., Downers Grove, IL) on archival cytologic preparations, we studied 23 patient samples, one Papanicolaou-and one Diff-Quik-stained slide per case (46 slides), and two additional unstained slides (fresh ascitic fluids) as controls. Included in this study were nine ascitic fluids (four benign and five malignant), four malignant pleural fluids, three benign bladder washes, and seven malignant fine-needle aspirates (FNA) from various sites. The slides ranged from 1-94 days old. After removal of coverslips using xylene, all slides were destained in a series of alcohol and water washes. Pretreatment of slides with pepsin was followed by the in situ hybridization procedure. Two hundred cells per slide were evaluated for distinct separate signals. Results showed the following: 1) all slides were evaluable except for eight (8/46) which had either too few cells or enough cells but with faint signals, 2) the oldest sample showed distinct signals, 3) previously Diff-Quik-stained slides showed relatively better signals than Papanicolaou-stained slides, 4) samples less than a month old showed relatively better signals, and 5) malignant samples showed various NCA, but not the benign samples. We conclude that FISH on archival cytologic preparation 1) is feasible, although age of the slide is a factor since better signals were seen in those less than a month old, 2) shows better results in previously Diff-Quik-stained slides, and 3) is a tool that can be used in the retrospective study of various liquid and solid neoplasms.

Additional observations on the preparation of R banded human chromosomes with acridine orange.

A number of technical factors which affect acridine orange R banding (RFA banding) were studied. These variables included age of slide, timing of fixation, details of incubation mounting and the use of sequential technics. Optimal RFA banding was obtained between 15 and 20 days but good or very good preparations were obtained between 7 days and 2 months. Improved results were obtained in slides that were 3-4 months old by refixing the slides in ethanol acetic acid. Intermittent movement of slides during incubation in buffer as well as the details of mounting and removal of cover slips were found to be important. The best sequential banding was obtained with the sequence of Q to R but good results were obtained with the sequence G to R using ASG banding. Satisfactory results with the sequence R to C were not obtained. With careful attention to these variables good RFA binding can be obtained over a period of several months.

Use of Ear Chamber with Uterine Muscle Transplant to differentiate Myometrial and Vascular Activity

THE Sandison1,2–Clark3 rabbit ear chamber technique has been used extensively for the study of mammalian tissues in vivo. The type of ear chamber used in the present investigation4 was constructed mainly of ‘Perspex’ as in the Sandison–Clark chamber but has been adapted for removal of coverslip for implantation of grafts after the method of Williams and Roberts5. It has been shown that within such grafts the vasculature of the donor organ persists6.