Quantification of Neuronal Dendritic Spine Density and Lengths of Apical and Basal Dendrites in Temporal Lobe Structures Using Golgi-Cox Staining.

The objective of this chapter is to provide an overview of the methods used to investigate the connectivity and structure of the nervous system. These methods allow neuronal cells to be categorized according to their location, shape, and connections to other cells. The Golgi-Cox staining gives a thorough picture of all significant neuronal structures found in the brain that may be distinguished from one another. The most significant characteristic is its three-dimensional integrity since all neuronal structures may be followed continuously from one part to the next. Successions of sections of the brain's neurons are seen with the Golgi stain. The Golgi method is used to serially segment chosen brain parts, and the resulting neurons are produced from those sections.

Development of an image processing software for quantification of histological calcification staining images.

Quantification of the histological staining images gives important insights in biomedical research. In wet lab, it is common to have some stains off the target to become unwanted noisy stains during the generation of histological staining images. The current tools designed for quantification of histological staining images do not consider such situations; instead, the stained region is identified based on assumptions that the background is pure and clean. The goal of this study is to develop a light software named Staining Quantification (SQ) tool which could handle the image quantification job with features for removing a large amount of unwanted stains blended or overlaid with Region of Interest (ROI) in complex scenarios. The core algorithm was based on the method of higher order statistics transformation, and local density filtering. Compared with two state-of-art thresholding methods (i.e. Otsu's method and Triclass thresholding method), the SQ tool outperformed in situations such as (1) images with weak positive signals and experimental caused dirty stains; (2) images with experimental counterstaining by multiple colors; (3) complicated histological structure of target tissues. The algorithm was developed in R4.0.2 with over a thousand in-house histological images containing Alizarin Red (AR) and Von Kossa (VK) staining, and was validated using external images. For the measurements of area and intensity in total and stained region, the average mean of difference in percentage between SQ and ImageJ were all less than 0.05. Using this as a criterion of successful image recognition, the success rate for all measurements in AR, VK and external validation batch were above 0.8. The test of Pearson's coefficient, difference between SQ and ImageJ, and difference of proportions between SQ and ImageJ were all significant at level of 0.05. Our results indicated that the SQ tool is well established for automatic histological staining image quantification.

A CNN-based approach for joint segmentation and quantification of nuclei and NORs in AgNOR-stained images.

BACKGROUND AND OBJECTIVE: Oral cancer is the sixth most common kind of human cancer. Brush cytology for counting Argyrophilic Nucleolar Organizer Regions (AgNORs) can help early mouth cancer detection, lowering patient mortality. However, the manual counting of AgNORs still in use today is time-consuming, labor-intensive, and error-prone. The goal of our work is to address these shortcomings by proposing a convolutional neural network (CNN) based method to automatically segment individual nuclei and AgNORs in microscope slide images and count the number of AgNORs within each nucleus. METHODS: We systematically defined, trained and tested 102 CNNs in the search for a high-performing solution. This included the evaluation of 51 network architectures combining 17 encoders with 3 decoders and 2 loss functions. These CNNs were trained and evaluated on a new AgNOR-stained image dataset of epithelial cells from oral mucosa containing 1,171 images from 48 patients, with ground truth annotated by specialists. The annotations were greatly facilitated by a semi-automatic procedure developed in our project. Overlapping nuclei, which tend to hide AgNORs, thus affecting their true count, were discarded using an automatic solution also developed in our project. Besides the evaluation on the test dataset, the robustness of the best performing model was evaluated against the results produced by a group of human experts on a second dataset. RESULTS: The best performing CNN model on the test dataset consisted of a DenseNet-169 + LinkNet with Focal Loss (DenseNet-169 as encoder and LinkNet as decoder). It obtained a Dice score of 0.90 and intersection over union (IoU) of 0.84. The counting of nuclei and AgNORs achieved precision and recall of 0.94 and 0.90 for nuclei, and 0.82 and 0.74 for AgNORs, respectively. Our solution achieved a performance similar to human experts on a set of 291 images from 6 new patients, obtaining Intraclass Correlation Coefficient (ICC) of 0.91 for nuclei and 0.81 for AgNORs with 95% confidence intervals of [0.89, 0.93] and [0.77, 0.84], respectively, and p-values < 0.001, confirming its statistical significance. Our AgNOR-stained image dataset is the most diverse publicly available AgNOR-stained image dataset in terms of number of patients and the first for oral cells. CONCLUSIONS: CNN-based joint segmentation and quantification of nuclei and NORs in AgNOR-stained images achieves expert-like performance levels, while being orders of magnitude faster than the later. Our solution demonstrated this by showing strong agreement with the results produced by a group of specialists, highlighting its potential to accelerate diagnostic workflows. Our trained model, code, and dataset are available and can stimulate new research in early oral cancer detection.

Silver Nitrate Staining of Nucleolar Organizer Regions (Ag-NORs) in Plant Chromosomes.

Silver nitrate staining to evidence the location of nucleolar organizer regions (Ag-NORs) in chromosomes is widely used as a classical method in plant cytogenetics. Here, we present the most used procedures and highlight some aspects in terms of their replicability by plant cytogeneticists. Some technical features described are materials and methods used, procedures, protocol modifications, and precautions in order to obtain positive signals. The methods to obtain Ag-NOR signals have different degrees of replicability, but do not require any sophisticated technology or equipment for their application.

A QCM immunosensor employing signal amplification strategies by enlarging the size of nanoparticles using gold or silver staining.

We present quartz crystal microbalance (QCM) immunosensors for the detection of alpha-fetoprotein (AFP) in a human serum immunoassay with high sensitivity. In this study, we employed three types of signal amplification strategies using size enlargement and/or increase in mass of gold and titanium dioxide nanoparticles. Since the basic principle of the QCM sensor is to measure the change in resonance frequency according to the mass change caused by the molecular interactions on the sensor surface, we were able to quantitatively analyze AFP by sandwich immunoassay using gold or titanium dioxide nanoparticles conjugated with anti-AFP detection antibodies and the subsequent three signal amplification techniques in a similar manner. The signal amplification technologies provide the size expansion of gold nanoparticles by gold or silver staining reaction and mass enhancement by photocatalytic silver staining of titanium dioxide nanoparticles. The limit of detections (LODs) of the AFP immunoassay in human serum by the gold and silver staining-mediated signal amplifications for gold nanoparticles were 56 and 87 pg mL-1, respectively, but by the photocatalytic silver staining signal amplification for titanium dioxide nanoparticles was 118 pg mL-1. This means that the signal amplification method through size enhancement by gold staining of gold nanoparticles further improved the detection ability of the QCM immunosensor.

Could Argyrophilic Nucleolar Organizer Regions count mirror DNA Ploidy in Malignant Salivary Gland Tumors?

OBJECTIVE: Nucleolar organizer regions (NORs) are DNA coils that transcribe to ribosomal RNA. The NOR-associated protein, termed argyrophilic NOR (AgNOR), was visible within the nucleus by staining with silver nitrate examination via the light microscope. AgNOR counting is a proliferation marker and may help in the diagnosis and prognosis of various neoplastic lesions. Aneuploidy (abnormal DNA content) can predict the progression, survival and prognosis of the tumors. The aim of this study was to evaluate the role of AgNORs, DNA ploidy status, and total S-phase fraction (TSPF) as prognostic parameters in malignant salivary gland tumors (MSGTs). METHODS: The current study is a retrospective study on a cohort of MSGTs (N=47), to assess AgNORs using Silver Nitrate stain, DNA index (DI), and TSPF using flow cytometry (FCM). Data including tumor size and site, lymphovascular invasion (LVI), lymph node metastasis (LNM) were collected. RESULTS: The AgNORs count was statistically significant with MSGT type. DI was found to have a significant association with tumor site, tumor size and MSGT type. In addition, TSPF was found to be significantly associated with LVI. A moderate positive correlation was noted between AgNORs count and TSPF. LNM, tumor site, high AgNORs and low DI were all associated with short disease-free survival (DFS) and poor overall survival (OS). CONCLUSION: The present study revealed that high AgNORs count, DNA aneuploidy and TSPF had a poor influence on MSGTs prognosis.

Improvement of the silver staining method for bacterial flagella.

Flagella staining is a common method used in microbial research to identify and mark morphological features of bacteria. We improved the Blendon staining method by adding two steps to the usual procedure, viz. "preparation of a pre-atomized microscope slide" and "stretching flagella in situ". The staining effects were then comparatively studied for this new, improved method on Bacillus subtilis, under four different culture conditions: 1) liquid culture medium, 2) aqueous solution at the bottom of slant medium, 3) solid culture medium adding water for stretching after culture, and 4) semi-solid culture medium adding water for stretching after culture. The results revealed that after the addition of these two steps to the usual procedure, the order of the staining effects for the four culture conditions from best to worst was as follows: semi-solid culture medium > solid culture medium > aqueous solution at the bottom of slant medium > liquid culture medium. Hence, the semi-solid culture medium brought about the best staining effect, with flagella stretching freely and not entangled with each other, while the liquid culture medium had the worst staining, owing to the serious background interference. This improved method is simple, low cost, and worthy of promotion.

Modification in Silver Staining Procedure for Enhanced Protein Staining.

Silver staining is an excellent technique for detecting proteins that are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein silver staining technology has higher sensitivity and is suitable for the detection of low-concentration proteins compared to other staining techniques including the Coomassie brilliant blue detection method. The present study was conducted to enhance the detection ability of the protein staining method. Herein, we modified the recipe of silver staining, a very reproducible method, by adding AMP, PVP, Tween-80, and xylene to enhance the detection ability of protein staining. Furthermore, the particle size and potentiometer were used to detect the particle size and potential difference of the silver ions in the prepared dyeing materials, and then, the morphology, transparency, and size of the dyed silver particles in different dyeing solutions were studied using a transmission electron microscopy (TEM). The obtained results revealed that the use of 0.5% of AMP, PVP, Tween-80, and xylene improved the staining ability of protein silver staining, compared with the original method. Furthermore, 0.5% AMP, 0.5% PVP, 0.5% Tween-80 reagents significantly influenced the morphology, size, potential, and dispersion of silver ions. These results suggested a new idea for further improving the detection ability of protein silver staining.

Golgi Silver Staining of Mosquito Central Neuropils.

The Golgi silver staining procedure relies on three interdependent stages: fixation, chromation, and metal impregnation. Each of these stages can be modified. This protocol describes a method demonstrated to stain neurons within the mosquito central nervous system. The resulting preparations are stable at room temperature.

The Golgi Method.

Silver staining by the Golgi method is a classical procedure to identify the three-dimensional morphology of individual neurons. Although the method was developed in the 1870s, it is still used to study the morphological characteristics of neuron types in the central nervous system, either alone or in combination with other neuroanatomical techniques. The neuropil of insects is fully accessible to the original refractive staining procedure, and modifications of the Golgi technique have paved the path for modern structural research on the insect central nervous system. Here, we provide an introduction to this easy and low-cost method.

Adapted technique for demonstrating argyrophilic nucleolar organizer regions in the cytologic samples of canine mast cell tumors.

INTRODUCTION: Argyrophilic nucleolar organizer regions (AgNORs) are nonhistone argyrophilic nucleolar proteins associated with ribosomal genes found in the nucleolar organizer region that reflect cell proliferation and have an affinity for silver. AgNOR staining may be useful to evaluate prognosis in several neoplasms because higher AgNOR counts are related to higher grade tumors, metastases, and shorter survival times. OBJECTIVE: We aimed to report on a quick and practical technique to identify AgNORs adapted for use in routine cytology. MATERIALS AND METHODS: The cytopathologic diagnosis of mast cell tumor (MCT) in samples collected by fine-needle aspiration (FNA) was determined. Next, slides were impregnated with a solution containing silver nitrate; the main modification of our technique included incubation of these slides at a controlled temperature of 25 °C. Some slides were previously stained with Diff-Quik and others were only fixed with methanol. The slides were analyzed under a microscope, and the number of blackened intranuclear points (AgNORs) was counted. RESULTS: Slides prestained with Diff-Quik were easily counted compared with slides only fixed in methanol. Technical issues encountered with the methanol-fixed slides included insufficient cellularity, background precipitation, and an absence of silver impregnation. CONCLUSIONS: The technique reported in this study showed satisfactory results for AgNOR counting in cytologic smears from MCT, such as good impregnation and the elimination of background interferents. Further evaluation of this method comparing AgNOR counts with histologic examinations, tumor grades, other prognostic markers, and survival times are needed to fully evaluate the benefit of this technique.

Comparative Study of Modified Silver Nitrate Staining for the Detection of Helicobacter pylori.

BACKGROUND/AIMS: Helicobacter pylori (Hp) infection is associated with a variety of diseases, such as benign lesions, precancerous lesions, and malignant lesions, especially diseases in the digestive system. Most people with Hp infection have mild early symptoms that are not easily noticed. Therefore, the diagnosis and treatment of Hp infection is particularly important. At present, there are many methods to detection Hp infection, but there is a lack of effective detection method with high sensitivity and specificity. On the basis of the existing detection methods, the modified silver nitrate staining method in this study improved the sensitivity and specificity of Hp detection. MATERIALS AND METHODS: We selected gastric antrum and gastric angle mucosal biopsy tissues from 60 inpatients that were archived in the Pathology Department of Zhongnan Hospital of Wuhan University from July to December 2020. An Hp immunohistochemical assay, histochemical assay kit (methylene blue), and modified silver nitrate staining were used to measure the Hp infection positivity rate. RESULTS: Comparison of Hp sensitivity and specificity among the 3 methods showed that the modified silver nitrate staining method was the most excellent. The sensitivity of modified silver nitrate staining method was 98.3%, which is statistically significantly higher compared with the other 2 methods. CONCLUSION: The modified silver nitrate staining method for Hp detection is convenient and effective, and could be widely used for clinical Hp detection.

Cytological Screening Model of Normal Oral Mucosa Exposed to Carcinogens: A Pilot Study.

INTRODUCTION: Oral cytopathology is able to detect incipient cellular alterations, but it is not routinely applied to this purpose. We aimed to establish a model to screen individuals with no oral lesion exposed to smoking/alcohol, by means of the nuclear area, cell proliferation rate, and analysis of genetic damage. METHODS: In this cross-sectional pilot study, 90 patients were allocated into 3 groups: oral cancer group (patients with oral squamous cell carcinoma), tobacco/alcohol group (patients without oral lesions and exposed to these risk factors), and control group (individuals with no lesion and not exposed to tobacco and alcohol). The cytological smears performed in these individuals were stained with Papanicolaou, a silver-staining and a Feulgen reaction. The nuclei of cells were measured, and AgNORs/nucleus and micronuclei (MN) were quantified. The cutoff values were stipulated evaluating the healthy mucosa (control group) and the cancerization field mucosa (oral cancer group). RESULTS: Cutoff values for the screening of individuals exposed to carcinogens were ≥8% of nuclei larger than 100 µm2, ≥3.38 AgNOR/nucleus, and ≥3 MN per 1,000 cells. CONCLUSIONS: Nuclear area measurement and AgNORs/nucleus and MN quantification identified the incipient phase of oral carcinogenesis. A screening model for individuals without oral lesion exposed to smoking/alcohol was proposed.

Staining Sodium Dodecyl Sulfate-Polyacrylamide Gels with Silver Salts.

This protocol describes silver staining procedures to detect low-abundance proteins in sodium dodecyl sulfate-polyacrylamide gels.

Whole-organism 3D quantitative characterization of zebrafish melanin by silver deposition micro-CT.

We previously described X-ray histotomography, a high-resolution, non-destructive form of X-ray microtomography (micro-CT) imaging customized for three-dimensional (3D), digital histology, allowing quantitative, volumetric tissue and organismal phenotyping (Ding et al., 2019). Here, we have combined micro-CT with a novel application of ionic silver staining to characterize melanin distribution in whole zebrafish larvae. The resulting images enabled whole-body, computational analyses of regional melanin content and morphology. Normalized micro-CT reconstructions of silver-stained fish consistently reproduced pigment patterns seen by light microscopy, and further allowed direct quantitative comparisons of melanin content across wild-type and mutant samples, including subtle phenotypes not previously noticed. Silver staining of melanin for micro-CT provides proof-of-principle for whole-body, 3D computational phenomic analysis of a specific cell type at cellular resolution, with potential applications in other model organisms and melanocytic neoplasms. Advances such as this in whole-organism, high-resolution phenotyping provide superior context for studying the phenotypic effects of genetic, disease, and environmental variables.

Optimization and application of silver staining of non-glycosylated and glycosylated proteins and nucleic acids for agarose native gel electrophoresis.

Electrophoresis is one of the major techniques to analyze macromolecular structure and interaction. Its capability depends on the sensitivity and specificity of the staining methods. We have here examined silver staining of proteins and nucleic acids separated by agarose native gel electrophoresis. By comparing five commercial kits, we identified Silver Stain Plus from Bio-Rad most adequate, as it provided little background staining and reasonable band staining. One of the disadvantages of the Silver Stain Plus kit is its variable staining of glycoproteins as tested with several model samples, including hen egg white proteins, α1-acid glycoprotein and SARS-CoV-2 Spike protein. One of the advantages of silver staining is its ability to stain nucleic acids as demonstrated here for a model nucleic acid with two kits. It was then used to monitor the removal of nucleic acids from the affinity-purified maltose binding protein and monoclonal antibody. It also worked well on staining proteins on agarose gels prepared in the vertical mode, although preparation of the vertical agarose gels required technological modifications described in this report. With the silver staining method optimized here, it should be possible in the future to analyze biological samples that may be available in limited quantity.

Silver Staining of 2D Electrophoresis Gels.

Silver staining is used to detect proteins after electrophoretic separation on polyacrylamide gels. It combines excellent sensitivity (in the low nanogram range) with the use of very simple and cheap equipment and chemicals. For its use in proteomics, two important additional features must be considered, compatibility with mass spectrometry and quantitative response. Both features are discussed in this chapter, and optimized silver staining protocols are proposed.

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining.

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role in a variety of basic biological structures and processes. As polymers, GAGs exist as a polydisperse mixture containing polysaccharide chains that can range from 4000 Da to well over 40,000 Da. Within these chains exists domains of sulfation, conferring a pattern of negative charge that facilitates interaction with positively charged residues of cognate protein ligands. Sulfated domains of GAGs must be of sufficient length to allow for these electrostatic interactions. To understand the function of GAGs in biological tissues, the investigator must be able to isolate, purify, and measure the size of GAGs. This report describes a practical and versatile polyacrylamide gel electrophoresis-based technique that can be leveraged to resolve relatively small differences in size between GAGs isolated from a variety of biological tissue types.

Reduced ribosomal DNA transcription in the prefrontal cortex of suicide victims: consistence of new molecular RT-qPCR findings with previous morphometric data from AgNOR-stained pyramidal neurons.

Prefrontal cortical regions play a key role in behavioural regulation, which is profoundly disturbed in suicide. The study was carried out on frozen cortical samples from the anterior cingulate cortex (dorsal and ventral parts, ACd and ACv), the orbitofrontal cortex (OFC), and the dorsolateral cortex (DLC) obtained from 20 suicide completers (predominantly violent) with unknown psychiatric diagnosis and 21 non-suicidal controls. The relative level of ribosomal RNA (rRNA) as a marker of the transcriptional activity of ribosomal DNA (rDNA) was evaluated bilaterally in prefrontal regions mentioned above (i.e. in eight regions of interest, ROIs) by reverse transcription and quantitative polymerase chain reaction (RT-qPCR). The overall statistical analysis revealed a decrease in rDNA activity in suicide victims versus controls, particularly in male subjects. Further ROI-specific post hoc analyses revealed a significant decrease in this activity in suicides compared to non-suicides in five ROIs. This effect was accentuated in the ACv, where it was observed bilaterally. Our findings suggest that decreased rDNA transcription in the prefrontal cortex plays an important role in suicide pathogenesis and corresponds with our previous morphometric analyses of AgNOR-stained neurons.

A visual bio-barcode immunoassay for sensitive detection of triazophos based on biochip silver staining signal amplification.

Herein, a novel visual method for detecting triazophos based on competitive bio-barcode immunoassay was described. The competitive immunoassay was established by gold nanoparticles (AuNPs), magnetic microparticle (MMPs) and triazophos, combined with biochip hybridization system to detect the residual of triazophos in water and apple. Because AuNPs carried many bio-barcodes, which hybridized with labeled DNA on the biochip, catalyzed signal amplification using silver staining was detected by grayscale values as well as the naked eye. Notably, the grayscale values decreases with increasing the concentrations of triazophos, and the color change weakened gradually. The detection range was in between 0.05 and 10 ng/mL and the minimum detection limit was set at 0.04 ng/mL. Percent recovery calculated from spiked water and apple samples ranged between 55.4 and 107.8% with relative standard deviations (RSDs) of 12.4-24.9%. It has therefore been shown that this protocol provides a new insight for rapid detection of small molecule pesticides in various matrices.