Single Staining Method Research Articles

Semi-quantitative scoring criteria based on multiple staining methods combined with machine learning to evaluate residual nuclei in decellularized matrix

Abstract The detection of residual nuclei in decellularized extracellular matrix (dECM) biomaterials is critical for ensuring their quality and biocompatibility. However, current evaluation methods have limitations in addressing impurity interference and providing intelligent analysis. In this study, we utilized four staining techniques—HE staining, acetocarmine staining, the Feulgen reaction, and DAPI staining—to detect residual nuclei in dECM biomaterials. Each staining method was quantitatively evaluated across multiple parameters, including area, perimeter, and grayscale values, to establish a semi-quantitative scoring system for residual nuclei. These quantitative data were further employed as learning indicators in machine learning models designed to automatically identify residual nuclei. The experimental results demonstrated that no single staining method alone could accurately differentiate between nuclei and impurities. In this study, a semi-quantitative scoring table was developed. With this table, the accuracy of determining whether a single suspicious point is a cell nucleus has reached over 98%. By combining four staining methods, false positives caused by impurity contamination were eliminated. The automatic recognition model trained based on nuclear parameter features reached the optimal index of the model after several iterations of training in 172 epochs. The trained artificial intelligence model achieved a recognition accuracy of over 90% for detecting residual nuclei. The use of multi-dimensional parameters, integrated with machine learning, significantly improved the accuracy of identifying nuclear residues in dECM slices. This approach provides a more reliable and objective method for evaluating dECM biomaterials, while also increasing detection efficiency.

MiR-128-3p inhibits the proliferation, migration and invasion of human nasopharyngeal carcinoma cells via EMT

Purpose: To investigate the effect of overexpressing miR-128-3p on the migration, invasion, and proliferation of human nasopharyngeal cancer cells, and associated pathways.Methods: Cell counting kit-8 (CCK-8) and clone creation tests were used to investigate the effect of miR-128-3p inhibitor on the growth of C666-1 cells. The inhibitor control and miR-128-3p inhibitor were transfected into human nasopharyngeal cancer cells (C666-1) and allowed to incubate for 48 h. Cell migration and invasion assays were conducted using Transwell and Scratch assays. Cell cycle was assessed using propidium iodide (PI) single-staining method, while expression of miR-128-3p was evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Expression levels of vimentin, E-cadherin, and N-cadherin were determined by Western blot.Results: MiR-128-3p inhibitor transfection in C666-1 cells significantly increased cell proliferation, cell viability, and clonal proliferation, and also significantly reduced miR-128-3p expression levels, and enhanced cell migration and invasion. In C666-1 cells, inhibition of miR-128-3p induced epithelial mesenchymal transition (EMT).Conclusion: MiR-128-3p inhibits nasopharyngeal cancer cells from proliferating, migrating, and invading by regulating epithelial mesenchymal transition. MiR-128-3p may therefore be a viable therapeutic target for nasopharyngeal cancer. Future studies are required to yield pertinent data in support of this hypothesis.

Cytological diagnosis of exercise-induced pulmonary haemorrhage: Comparison of tracheal wash and bronchoalveolar lavage in standardbred racehorses.

Cytology of airway samples is sensitive for diagnosis of exercise-induced pulmonary haemorrhage (EIPH), but the association between tracheal wash (TW) and bronchoalveolar lavage fluid (BALF) is unknown. The objective of this study was to determine whether diagnosis of EIPH, using haemosiderophages/macrophages (H/M) ratio, differs when based on TW or BALF. A prospective cross-sectional study was conducted on 102 standardbred horses in training. TW and BALF were collected concomitantly from all horses at rest (at least 24hours after their last training or race), and their H/M ratios were calculated. Spearman's correlation, Cohen's kappa and Gwet's coefficient tests were performed to evaluate the association between TW and BALF samples. With BALF, 21 horses met the cytological inclusion criteria for an EIPH diagnosis from individual and/or pooled samples. With TW, 20 horses had occasional (H/M < 10%) haemosiderophages, and nine, one and three horses had small (10%-25%), moderate (25%-50%) and large (>50%) proportions, respectively. Poor correlations and inconsistent concordances between TW and BALF were found for H/M ratio. Limitations include the use of a single staining method and the absence of a total haemosiderin score. No association between TW and BALF was found for the cytological diagnosis of EIPH. Based on H/M ratio, BALF remains the sample type of choice for cytological diagnosis of EIPH.

Single-staining flow cytometry approach using SYTOX™ green to describe electroporation effects on Escherichia coli

Pulsed electric fields (PEF) can induce reversible or irreversible electroporation effects on bacterial cells resulting in sublethally or lethally injured cells. Hereafter, an alternative single-staining flow cytometry approach with SYTOX™ Green (SYTOX) is proposed, allowing for a straightforward and rapid screening of electroporation effects. SYTOX-staining indicated 38–63% of E. coli cells with intermediate cellular changes, likely being sublethally injured, at electric field intensities of 8 kV/cm–18 kV/cm. Compared to staining with propidium iodide, SYTOX showed a brighter fluorescence intensity, allowing for easier differentiation of subpopulations. The proportion of intermediate injury observed with SYTOX was distinctly higher than the sublethal damage detected with conventional plating on selective media (31–45%). It is, however, challenging to compare selective plating with the single-staining method based on membrane integrity, suggesting different analytical methods to indicate complex cellular states and sublethal effects.

Inactivation of specific spoilage organism (Pseudomonas) of sturgeon by curcumin-mediated photodynamic inactivation.

The present study aimed to measure the inactivation effect and mechanism of curcumin-mediated photodynamic inactivation (PDI) on the specific spoilage organism (Pseudomonas) of the sturgeon. The conditions of PDI used were as follows: 30 μM curcumin, 15 W LED light (470 nm) power and 90 s irradiation time. Under these conditions, the high-throughput sequencing was used to study the microbiota of sturgeon. The method of aerobic plate colony count (APC) was used to determine the viability of Pseudomonas after PDI. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the propidium iodide (PI) single staining method, and agarose gel electrophoresis were used to study the inactivation mechanism of PDI on Pseudomonas. The results showed that Pseudomonas was the specific spoilage organism of sturgeon, and PDI significantly inhibited the growth of Pseudomonas. The in-vitro inactivation rate of Pseudomonas was 99.9% with counts decreased by 3.19 ± 0.15 log10 CFU/mL. The mechanism of PDI to inactivate Pseudomonas is as follows. Firstly, the high-level structure of membrane protein was destroyed, and the cell membrane permeability was increased, which caused leakage of cellular content. Then the nucleic acid inside the cell was destroyed, which eventually caused the death of bacteria. These findings demonstrate that curcumin-mediated PDI can be utilized as an effective way to inactivate the specific spoilage organism (Pseudomonas) of the sturgeon.

AMMECR1 Inhibits Apoptosis and Promotes Cell-cycle Progression and Proliferation of the A549 Human Lung Cancer Cell Line.

The aim of this study was to characterize the role of Alport syndrome, mental retardation, midface hypoplasia, and elliptocytosis chromosomal region gene 1 (AMMECR1) in human lung cancer cell lines. AMMECR1 gene expression was evaluated in four lung cell lines, with A549 then selected for further in-depth examination. To characterize the role of AMMECR1, silencing was achieved utilizing lentivirus-mediated RNA interference, and confirmed by quantitative real-time polymerase chain reaction and western blotting. The impact of AMMECR1 silencing on cellular proliferation was assessed using Celigo-based and MTT assays. Apoptosis was determined using the annexin V-allophycocyanin single staining method. Cell-cycle arrest was assessed by flow cytometry. Finally, colony formation was assessed using Giemsa staining. In A549 cells, AMMECR1 silencing was found to significantly suppress cell proliferation, reduce colony formation, promote apoptosis, and arrest cells in the S and G2/M phases. AMMECR1 plays a critical role in cell proliferation, cell-cycle progression, and apoptosis of human lung cancer cells, and may serve as a potential therapeutic target for non-small-cell lung cancer.

Rapid whole blood assay using flow cytometry for measuring phagocytic activity of chicken leukocytes

Phagocytic activity of leukocytes in whole blood was assessed as a potential immune competence trait in chickens. A flow cytometry based whole blood phagocytosis (WBP) assay was set up and evaluated using blood from chickens homozygous for four different MHC haplotypes, B12, B15, B19 and B21. Fluorescent latex beads and two serotypes of fluorescently labelled heat-killed bacteria (Salmonella Infantis and Salmonella. Typhimurium) were evaluated as phagocytic targets. In addition, the opsonophagocytic potential (OPp) of individual sera from the birds was included in a phagocytosis assay using the HD11 chicken macrophage cell line. Results showed that both serotypes of bacteria but not the latex beads were effectively phagocytosed by leukocytes in the whole blood cultures. Differences were observed in the phagocytic capacity of monocytes and thrombocyte/lymphocytes, respectively between the different MHC lines. No significant differences on the OPp of serum was identified between MHC lines. In addition, for both phagocytic activity of leukocytes and OPp of serum large variations between individuals were observed within MHC haplotypes. No significant relationships were observed between the phagocytic activity of leukocytes and serum OPp or Salmonella-specific IgY levels.In conclusion, our results suggest that the WBP assay, using a no-lyse no-wash single staining method, is a rapid and convenient method to assess phagocytic functions of different leukocyte populations.

舌扁平上皮癌に対するヨード2回染色法の有用性の検討

We examined the usefulness of a double-staining method using iodine solutions of different concentrations for the excision of T1, T2 squamous cell carcinomas （SCCs） of the tongue. We studied 127 patients who were given a diagnosis of T1, T2 SCCs of the tongue at Maxillofacial Surgery, Tokyo Medical and Dental University from 2000 through 2010. The patients were classified into 2 groups. One group, consisting of 89 cases, underwent staining once with 3% iodine solution （single-staining group: Group I）. The other group, consisting of 38 cases, underwent staining with 3% iodine solution and then with 5% iodine solution （double-staining group: Group II）. The excision margin of the lesion was designed to be about 10 mm outside of the border of the unstained areas （USAs） and stained areas （SAs）. Then the lesion was excised. In addition, the borders of the SAs and USAs were examined, and any recurrence after excision was noted. The type of lesion, type of epithelial dysplasia at the surgical margin, and recurrence rate were compared between the two groups. Local recurrence and the presence of epithelial dysplasia at the surgical margin in SCCs cases were seen in 12/127 cases （9.4%） and 58/127 cases （45.7%）, respectively. The numbers of the surgical marginpositive cases were 47/89 cases （52.8%） in Group I and 11/38 （28.9%） in Group II. The rate of SCC recurrence was significantly lower in Group II than in Group I （p=0.01）. The number of cases with local recurrence was 12/89 （13.5%） in Group I, but 0/38 （0%） in Group II. These results suggest that the double-iodine-staining method indicated the border of epithelial dysplasia more clearly than the single-staining method. Therefore,the double-iodine-staining method is useful for determining the excision margin of SCCs of the tongue.

A novel method combining gene and protein platforms on one slide for the detection of HER2 in gastric cancer: Final results.

19 Background: The evaluation of gene amplification and/or protein overexpression of HER2 in gastric cancer is a prerequisite to establish an adequate treatment strategy. Gastric tumors are heterogeneous and separate evaluations lead to uncertainties in localizing distinct clones and are time consuming. The aim of this study was to evaluate the feasibility of gene-protein platform in comparison to single staining methods. Methods: Immunohistochemistry (IHC) plus silver in situ hybridization (SISH) (IHC/SISH) and the new gene-protein platform (gene/protein) method for HER2 were performed in randomly collected 100 cases of gastric carcinoma. Evaluation was performed by two observers, in discrepant cases a third observer was consulted to make a decision by consensus. Results of IHC and SISH were compared with gene/protein staining. Rüschoff criteria were applied. Tumors showing HER2 expression 3+ or amplification were considered HER2 positive. Results: 96 of 100 samples were eligible. Amplification was observed in 14.6% by both, conventional SISH and gene/protein platform. 70.8% by IHC and gene/protein had no expression (0) and 10.4%/11.5% (IHC vs. gene/protein) had weak (1+) HER2 expression. Moderate expression (2+) was observed in 9.4% by IHC and 7.3% by gene/protein. Rate of overexpression (3+) was similar in IHC (9.4%) and gene/protein (10.4%). There were complete concordances (100%; κ=1) in IHC assessment of cases with score 0 and SISH amplified tumors. High concordance are shown in score 1+ (98.96%; κ=0.947) and 3+ (96.88%; κ=0.825) cases. Concordance in cases with score 2+ was found in 95.83% (κ=0.728) of the observations. After third observation, there were 5 discordant cases with most discrepancies in assessment of IHC score 2+ or 3+. Conclusions: Gene-protein platform has been tested for first time in gastric carcinoma. Results showed that this novel platform can be a feasible alternative to single methods. Discrepancies in cases with moderate HER2 expression are a result of observer variability.

A comparison of immunohistochemistry (IHC) and dual-color silver in situ hybridization (SISH) with a novel method combining both gene and protein platforms on a unique slide for testing the HER2 in gastric carcinoma.

4100 Background: Amplification and/or protein overexpression of HER2 in gastric cancer is a prerequisite to establish an adequate treatment strategy. The European standard defined HER2 positivity by IHC as first evaluation assay followed by ISH in 2+ cases. Gastric tumors are heterogeneous and separate evaluations lead to uncertainties and in localizing distinct clones and are time consuming. The aim of this study was to evaluate the feasibility of gene-protein platform in comparison to single staining methods. Methods: IHC plus SISH and gene-protein platform (IHC/SISH, protein/gene) method for HER2 were performed in randomly collected 100 cases of gastric carcinoma. Results of IHC and SISH were compared with IHC/SISH staining. Rüschoff criteria were applied. Tumors were HER2 positive when expression 3+ or 2+ plus gene amplification (EU-Norm) was found. In Second definition (US-Norm), tumors showing HER2 expression 3+ or amplification were considered HER2 positive. Results: 96 of 100 samples were eligible. Amplification was observed in 14.6% and 15.6% by SISH and IHC/SISH. 71.9% by IHC vs. 75.0% by IHC/SISH had no expression (0) and 10.4% (IHC vs. IHC/SISH) had weak (1+) HER2 expression. Moderate expression (2+) and overexpression (3+) were observed in IHC 6.3%/11.5% and IHC/SISH 6.3%/8.3%, respectively. There were high concordances in IHC assessment of cases with score 0 (94.8%; κ=0.87) and 3+ (96.9%; κ=0.83) and moderate concordances in 1+/2+ cases (89.6%; κ= 0.44 vs. 93.8%; κ=0.47). Rate of HER2 positivity was similar in standard or novel method. In EU Definition 14.6% vs. 10.4% (p=0.52) were positive, respectively, with very good concordance (95.8%; κ=0.81).Concordance between HER2 positivity in standard or novel method was very good (99.0%; κ=0.96) in US definition with no significant differences (17.7% vs. 16.7%; p=1). Conclusions: Gene-protein platform has been tested for first time in gastric carcinoma. Results showed that this novel platform can be a feasible alternative to single methods. Discrepancies in cases with weak or moderate HER2 expression can be a result of observer variability.

Standardization of CD62P measurement: results of an international comparative study

Despite long being a mainstay in describing platelet activation via degranulation, interlaboratory variation remains an issue in measurement of membrane CD62P by flow cytometry. Our objective was to identify actions that may minimize this variation. Sixteen laboratories participated in an international comparative study. Two sets of platelet samples were prepared in one laboratory. Set 1 was stained and fixed; set 2 was fixed and required staining at participating laboratories. A single-staining method was used, and platelet populations were selected based on forward scatter/side scatter characteristics. Calibration beads were used to standardize measurement across different instruments. There was a large discrepancy in reported CD62P values among study sites [interlaboratory coefficient of variance (CV): 36-78%]. When electronic data were re-analysed by a single analyst using a consistent gating strategy and a stable reference point, variation decreased markedly (CV<12%), indicating a problem with isotype control samples, possibly related to sample fixation or shipment. Consensus regarding gating strategies and use of a reliable reference point would greatly improve agreement in interlaboratory CD62P measurement.

Rapid Image-based Cytometry for Comparison of Fluorescent Viability Staining Methods

The ability to accurately measure cell viability is important for any cell-based research. Traditionally, viability measurements have been performed using trypan blue exclusion method on hemacytometer, which allowed researchers to visually distinguish viable from nonviable cells. However, the trypan blue method is often limited to only cell lines or primary cells that have been rigorously purified. In the recent years, small desktop image-based cell counters have been developed for rapid cell concentration and viability measurement due to advances in imaging and optics technologies as well as novel fluorescent stains. In this work, we employed the Cellometer image-based cytometer to demonstrate the ability to simplify viability detection compared to the current methods. We compared various fluorescence viability detection methods using single- or dual-staining technique. Single-staining method using nucleic acid stains including ethidium bromide, propidium iodide, 7AAD, DAPI, Sytox Green and Sytox Red, and enzymatic stains including CFDA and Calcein AM were performed. All stains produced comparable results to trypan blue exclusion method for cell line samples. Dual-staining method using AO/PI, CFDA/PI, Calcein AM/PI and Hoechst 33342/PI that enumerates viable and non-viable cells was tested on primary cell samples with high debris contents. This method allowed exclusion of cellular debris and non-nucleated cells from analysis, which can eliminate the need to perform purification step during sample preparation, and improves the efficiency of viability detection method. Overall, these image-based fluorescent cell counters can simplify assay procedures as well as capture images for visual confirmation.

Location of PHM/VIP mRNA in human gastrointestinal tract detected by in situ hybridization

The expression of the gene for vasoactive intestinal polypeptide (VIP) and peptide histidine methionine (PHM) in the human gastrointestinal tract was studied by in situ hybridization and Northern blotting for PHM/VIP mRNA and immunocytochemistry using specific antisera against the bioactive peptides PHM and VIP. In the colon sigmoideum, antisera against all five putative processing products of the VIP precursor (prepro-VIP) were used, namely prepro-VIP 22-79, PHM, prepro-VIP 111-122, VIP and prepro-VIP 156-170. Furthermore, RNA extracted from various regions of the gastrointestinal tract was examined by Northern blots and hybridization to a VIP-cDNA probe. Throughout the gastrointestinal tract, PHM/VIP mRNA was found in neurons only. Using single- or double-staining methods, we demonstrated both PHM/VIP mRNA and the corresponding peptides PHM and VIP in the neurons. In the sigmoideum, the single-staining methods were extended to investigate whether the neurons simultaneously contained PHM/VIP mRNA and each of the five prepro-VIP-derived peptides. Only one major band of PHM/VIP mRNA (1.9 kb) was found by Northern blotting in the tissue of the gastrointestinal tract.

Improved detection ofPneumocystis carinii by an immunofluorescence technique using monoclonal antibodies

To assess whether a recently developed indirect immunofluorescent stain using monoclonal antibodies was more sensitive in detecting Pneumocystis carinii than the combination of Giemsa and methenamine silver nitrate stains which has routinely been used in the laboratory, 88 lavage fluid specimens and 34 induced sputum specimens were examined. All specimens were stained by five techniques: immunofluorescence using a combination of three monoclonal antibodies (from the National Institutes of Health, USA), immunofluorescence using a single monoclonal antibody (from Dakopatts), Giemsa, methenamine silver nitrate and toluidine blue O. Immunofluorescence using the monoclonal antibodies from the NIH was significantly more sensitive than any other single staining method and than the combination of Giemsa and methenamine silver nitrate staining. The study also showed that the cytospin centrifuge was very suitable for the preparation of slides with lavage fluid and processed induced sputum. Finally, the sensitivity of examination of induced sputum to detect Pneumocystis carinii was found to be 50% when compared with bronchoalveolar lavage fluid. However, this sensitivity may increase through practice.

Immunocytochemical localization of the posterior lobe hormones and of their carrier proteins.

Serial sections of vertebrate hypothalami were stained with the immunocytochemical peroxidase-antiperoxidase method. In addition to the single staining method, our double staining method was used, which enabled us to visualize two tissue antigens in single tissue sections. In both staining methods, differentially adsorbed antineurohypophysial hormone sera, anti-somatostatin serum and anti-bovine neurophysin sera were used. The results confirm the one hormone, one neuron hypothesis.

THE USE OF COLOPHONIUM IN DIFFERENTIATING THE EOSIN-METHYLENEBLUE AND OTHER STAINS

One of the most important of the regressive staining methods used for general histological work is the eosinmethylene-blue method, hitherto only applicable to tissues fixed in Zenker's fixative. A successful stain by this method yields better results for cell-differentiation and the study of fine structures than any other single staining method. Its use has been limited because of the difficulty of securing constant results. Inquiries made in various laboratories, and the results of personal experience in a number of laboratories, have shown that the one uncertain step in the staining method is that of differentiation. It has been generally known that alcohols from different sources do not act alike when used for differentiation. The result of an attempt to determine the variable ingredient in the commercial 95 per cent, alcohol has yielded a method for differentiation which has given uniform results over a period of one year with a large