Silver Staining Method Research Articles

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.

Diosgenin ameliorates cellular and molecular changes in multiple sclerosis in C57BL/6 mice

Multiple sclerosis (MS) is especially known as a demyelinating disease of the central nervous system. Current treatments for MS are mostly based on controlling neuroinflammation and there are no treatments to promote the remyelination process at present. Diosgenin is a known herbal anti-inflammatory and antioxidant agent, which has also been shown to stimulate the growth of myelin in vitro. However, there is no or little evidence about diosgenin effects; specially on myelination, neuroprotection and its corresponding mechanisms in vivo in experimental autoimmune encephalomyelitis (EAE) as the most valid experimental model of MS. In this study, the therapeutic effect of diosgenin on clinical signs of EAE, and the corresponding cellular and molecular mechanisms have been examined with emphasis on myelination and neuroprotection mechanisms. EAE was induced using myelin oligodendrocyte glycoprotein (MOG) antigen in C57BL/6 mice. Diosgenin was gavaged (100 mg/kg) daily with the onset of paralysis signs (half tail paralysis) until the 18th post-immunization day in the treatment group. Blood and spinal cord tissue sampling was performed on post-immunization day 18. Lumbar spinal cord inflammation, demyelination, and axonal degeneration were assessed using Hematoxylin and Eosin (H & E), Luxol Fast Blue (LFB), and Bielschowsky's silver staining methods, respectively. Serum and spinal cord tissue level of tumor necrosis factor alpha (TNFα) and tissue levels of matrix metalloproteinase 9 (MMP-9) and interleukin 17 (IL-17) as inflammatory markers, microtubule-associated proteins 1A/1B light chain 3A (MAP1LC3A), and activity dependent neuroprotector homeobox (ADNP) as neuroprotective markers were assayed using enzyme linked immunosorbent assay (ELISA) method. The clinical score of EAE in the diosgenin treatment group was significantly reduced compared to the EAE group on days 15 to 18 after induction of the EAE (p < 0.001). Inflammation, demyelination and axonal loss scores also decreased significantly in the diosgenin treatment group compared to the EAE group (p < 0.05). Serum and spinal cord tissue level of TNFα and tissue level of MMP-9 considerably decreased in the diosgenin treatment group in comparison with the EAE group (p < 0.01). Diosgenin treatment had no significant effects on the tissue levels of IL-17, ADNP and MAP1LC3A. Therefore, diosgenin improved the clinical signs of EAE through lowering neuroinflammation, demyelination and axonal degeneration, but did not significantly affect the neuroprotective factors in this study. As a result, diosgenin could be a good candidate for new MS treatment strategies that, in addition to their anti-inflammatory effects, also enhance myelination.

Morphology, morphogenesis and phylogeny of Anteglaucoma orientalis n. sp. (Ciliophora, Oligohymenophorea), a freshwater hymenostomatid from northeastern China

A new hymenostomatid ciliate, Anteglaucoma orientalis n. sp., isolated from a freshwater pond in Harbin, northeastern China, was investigated using live observation and silver staining methods. Anteglaucoma orientalis is characterized as follows: size in vivo about 50–60 × 30–35 μm; oval body shape; buccal area occupies about 25% of body length; 28–36 somatic kineties; membranelle 1 having six or seven basal body rows, membranelle 2 five to seven rows, and membranelle 3 three rows; single macronucleus with one micronucleus attached. Morphogenesis of the genus Anteglaucoma is revealed for the first time. The main events during binary fission are as follows: morphogenesis begins with proliferation of kinetosomes in the middle part of postoral kinety 1, and kinetosomes of this primordial field multiply and organize to finally form the paroral membrane and membranelles 1–3 of the opisthe; the parental apparatus in the proter does not take part in the stomatogenetic process. Phylogenetic analyses based on SSU rRNA gene sequences show that Anteglaucoma orientalis n. sp. clusters with the type species, A. harbinensis Pan et al., 2017, with full support.

Identification and infection mechanism of Tetrahymena vorax affecting the goldfish Carassius auratus

Tetrahymena vorax is the ciliate responsible for tetrahymenosis in the goldfish Carassius auratus. The morphology of parasitic Tetrahymena vorax isolated from C. auratus from an ornamental-fish market in Harbin was studied using living observation and silver staining methods. Carassius auratus had large, white or grey skin lesions located on various parts of the body but were most common laterally. Gills of all diseased fish were shrivelled, dark and had white ulceration. The eyes of several diseased fish had turned grey and their fins were damaged or partially blackened. Histopathological examination revealed a systemic infection, pathological features of C. auratus include presence of large, white or grey skin lesions, dark shrivelled gills, grey eyes, damaged and/or partially blackened fins and invasion of ciliate cells in epidermis and dermis, viscera and abdominal cavity. Successful systemic infection of C. auratus was achieved with T. vorax by three tested modes (scratch, immersion and injection). The results indicated that T. vorax invade C. auratus through lesions of the body surface of injured specimens.

Incorporating mitogenome sequencing into integrative taxonomy: The multidisciplinary redescription of the ciliate Thuricola similis (Peritrichia, Vaginicolidae) provides new insights into the evolutionary relationships among Oligohymenophorea subclasses

The evolutionary relationships among Oligohymenophorea subclasses are under debate as the phylogenomic analysis using a large dataset of nuclear coding genes is significantly different to the 18S rDNA phylogeny, and it is unfortunately not stable within and across different published studies. In addition to nuclear genes, the faster-evolving mitochondrial genes have also shown the ability to solve phylogenetic problems in many ciliated taxa. However, due to the paucity of mitochondrial data, the corresponding work is scarce, let alone the phylogenomic analysis based on mitochondrial gene dataset. In this work, we presented the characterization on Thuricola similis Bock, 1963, a loricate peritrich (Oligohymenophorea), incorporating mitogenome sequencing into integrative taxonomy. As the first mitogenome for the subclass Peritrichia, it is linear, 38,802 bp long, and contains two rRNAs, 12 tRNAs, and 43 open reading frames (ORFs). As a peculiarity, it includes a central repeated region composed of tandemly repeated A-T rich units working as a bi-transcriptional start. Moreover, taking this opportunity, the phylogenomic analyses based on a set of mitochondrial genes were also performed, revealing that T. similis, as a representative of Peritrichia subclass, branches basally to other three Oligohymenophorea subclasses, namely Hymenostomatia, Peniculia, and Scuticociliatia. Evolutionary relationships among those Oligohymenophorea subclasses were discussed, also in the light of recent phylogenomic reconstructions based on a set of nuclear genes. Besides, as a little-known species, T. similis was also redescribed and neotypified based on data from two populations collected from wastewater treatment plants (WWTPs) in Brazil and Italy, by means of integrative methods (i.e., living observation, silver staining methods, scanning and transmission electron microscopy, and 18S rDNA phylogeny). After emended diagnosis, it is characterized by: (1) the sewage habitat; (2) the lorica with a single valve and small undulations; (3) the 7–22 µm-long inner stalk; and (4) the presence of only a single postciliary microtubule on the left side of the aciliferous row in the haplokinety. Among Vaginicolidae family, our 18S rRNA gene-based phylogenetic analysis revealed that Thuricola and Cothurnia are monophyletic genera, and Vaginicola could be a polyphyletic genus.

Detection of two pathogenic marine ciliates Ancistrum haliotis and A. crassum (Ciliophora: Scuticociliatia) by fluorescence in situ hybridization

The scuticociliatid ciliates Ancistrum haliotis and A. crassum are parasites that may cause high mortality in the cultured abalone Haliotis spp. and the bivalve Ruditapes philippinarum. Traditional identification with silver staining methods is hampered by their morphological similarities to closely related species and the complicated procedures of morphological analysis. We designed two SSU rRNA-targeted oligonucleotide probes labeled with a fluorochrome, and optimized the fluorescence in situ hybridization (FISH) protocols for identification of A. halioti and A. crassum, respectively. The assays resulted in a clear identification by strong fluorescence signals from the oligonucleotide probes. The method can be used for quick and accurate quantitative analysis of A. haliotis and A. crassum infections on host molluscs.

Fabrication and characterisation of chitosan/polyvinyl alcohol-based transparent hydrogel films loaded with silver nanoparticles and sildenafil citrate for wound dressing applications

ABSTRACT The wounds caused by either burning or trauma are recognised potentially dangerous. Hydrogels are perceived as promising materials in wounds care applications. In the first step, various compounds were fabricated to obtain the optimal composition and then characterised using several techniques, including a water vapour passage test, antibacterial test, scanning electron microscopy, water absorption, tensile strength, biodegradability, and Fourier transform infrared spectroscopy. Next, silver nanoparticles (AgNPs) plus sildenafil citrate as a well-known angiogenesis stimulant were added to the compound. The prepared wound dressings with different compounds were applied to three Wistar rat groups. Haematoxylin and eosin, and silver staining methods were used. The results revealed the optimised sample and the sample possessing the AgNPs outperformed the others in antibacterial activity. The angiogenesis was significantly triggered by the sildenafil citrate-treated group, and the better recovery was observed for rats treated with the optimised sample encompassing AgNPs and sildenafil citrate.

D-serine Ameliorates Motor and Cognitive Impairments in β-amyloid 1-42 Injected Mice by Inhibiting JNK Signaling Pathway

The senile plaque formed by β-amyloid (Aβ) deposition in the brain is one of the main pathological features of Alzheimer’s disease (AD), and the c-Jun N-terminal kinase (JNK) signaling pathway plays an important role in the pathogenesis of AD. This study aimed to investigate that D-serine may ameliorate motor and cognitive impairment in Aβ injected mice by inhibiting JNK signaling pathway. Firstly, Kunming mice were injected intrahippocampally with Aβ1-42 to build AD model. The mice were injected intraperitoneally with saline, D-serine, D-amino acid oxidase (DAAO), and Sodium benzoate (BE) for 10 consecutive days, respectively. Subsequently, the motor and cognitive functions of mice were detected by behavioral tests. The silver staining and immunohistochemical methods were used to detect the distributions of Aβ in the hippocampus of mice. 18F-2-Fluro-D-deoxy-glucose positron emission tomography/computed tomography (18F-FDG PET/CT) scans were performed to detected glucose metabolism of Aβ1-42 induced lesions. The expressions of relative JNK factors were detected by immunohistochemistry and Western blot methods. These results showed that Aβ severely impaired the motor and memory abilities of mice. The expressions of glial fibrillary acidic protein (GFAP), tumor necrosis factor (TNF-α), N-methyl-D-aspartate receptor 1 (NMDAR1), phospho-JNK (p-JNK), p-c-Jun and activating transcription factor 2 (ATF2) increased significantly. After D-serine treatment, the abilities of movement and memory of mice were improved, and the clearance rate of Aβ was accelerated. The expressions of GFAP, TNF-α, NMDAR1, p-JNK, p-c-Jun and ATF2 decreased significantly. DAAO and BE were administered to further validate these results. Therefore, this study showed that D-serine could alleviate the cognitive impairment of Aβ1-42 injected mice by inhibiting JNK signaling pathway. These results provide more evidences for the effect of D-serine on AD and relevant mechanism to treat AD.

Genetic Diversity of Indonesian Swamp Buffalo Based on Microsatellite Markers

Indonesia has high genetic resources of local swamp buffalo with good adaptation across regions. However, these animals decline in both population and genetic quality. This research was conducted to study the genetic diversity of Indonesian swamp buffalo. A total of 199 DNA samples (swamp buffalo) from seven provincial populations were used in this study. Genetics identification used three microsatellite markers (CSSM66, ILSTS61, and ILSTS17). Microsatellites were visualized by Polyacrylamide Gel Electrophoresis (PAGE) 10% with silver staining method. Microsatellite data were analyzed using GenAlEx 6.41, Cervus 3.0, and POPTREE2 software. The results showed that a total of 9 alleles were found from the three loci. ILSTS61 had a high PIC (Polymorphism Information Content) compared to the other loci. The high observed heterozygosity of ILSTS61 was found in swamp buffalo from Riau Province, while the Ho value of ILSTS17 ranged from 0.000 to 0.170. This study identified two clusters for Indonesian swamp buffalo, i.e., cluster I (Aceh, North Sumatra, and Riau) and cluster II (Banten, Central Java, West Nusa Tenggara, and South Sulawesi). The two major divergent directions are considered in Indonesia swamp buffaloes across the observed provinces.

Impact of single nucleotide polymorphisms in the OGG1 and XRCC1 genes on modulation of DNA damage in pesticide-exposed agricultural workers in Punjab, North-West India

Pesticide-induced DNA damage is primarily repaired by base excision repair (BER) pathway. However, polymorphism in DNA repair genes may modulate individual’s DNA repair capacity (DRC) leading to increased genotoxicity and adverse health effects. Our first study in North-West Indian population aimed to evaluate the impact of OGG1 rs1052133 (Ser326Cys; C1245G), XRCC1 rs1799782 (Arg194Trp; C26304T) and XRCC1 rs25487 (Arg399Gln; G28152A) polymorphisms on the modulation of pesticide-induced DNA damage in a total of 450 subjects (225 pesticide-exposed agricultural workers and 225 age- and sex-matched controls). DNA damage was estimated by alkaline comet assay using silver-staining method. Genotyping was carried out by PCR-RFLP using site-specific restriction enzymes. Mann-Whitney U-test revealed elevation in DNA damage parameters (p < 0.01) in pesticide-exposed agricultural workers than controls. Chi-square test showed significant (p < 0.05) differences in the XRCC1 Arg194Trp (C26304T) and Arg399Gln (G28152A) genotypes among two groups. Multivariate logistic-regression analysis revealed that heterozygous genotypes of OGG1 rs1052133 (326Ser/Cys; 1245CA), XRCC1 rs1799782 (194Arg/Trp; 26304CT) and XRCC1 rs25487 (399Arg/Gln; 2815GA) were positively associated (p < 0.05) with elevated DNA damage parameters in pesticide-exposed agricultural workers. Our results strongly indicate significant positive association of variant OGG1 and XRCC1 genotypes with reduced DRC and higher pesticide-induced DNA damage in North-West Indian agricultural workers.

Staining the Blot for Total Protein with Ponceau S.

Before probing blots for the presence of an antigen, the total composition of the transferred proteins can be determined by staining the nitrocellulose or polyvinylidene fluoride (PVDF) membrane. Staining for proteins is useful to determine the position of the non-prestained molecular weight markers or individual lanes on the gel and to ensure that efficient transfer has occurred. It can be also used to verify equal loading of the samples in the gel when a comparison of the protein of interest between the different samples is important. The conventional procedures such as Coomassie Blue and silver staining methods used for staining polyacrylamide gels are incompatible with immunoblotting. Ponceau S is the more common staining method in immunoblotting protocols because it is compatible with antibody-antigen binding, is cost efficient, and provides a good contrast between the stained bands and background. In this protocol, nitrocellulose or PVDF membrane is rinsed with ultrapure H2O after the transfer of proteins. Ponceau S dye is applied as an acidic aqueous solution, and the proteins on the membrane are stained with red color. The membrane is briefly destained with water and can be photographed or scanned to obtain the image of the total protein staining. Individual lane positions or the molecular weight standards can be marked with a pencil, if required.

Histochemical and immunohistochemical detection of pathogenic leptospires serovars in tissues of some captive wildlife from a University zoo in Nigeria

ABSTRACTThe detection and documentation of pathogenic Leptospira serovars in wild captive and zoological garden animals are scarce in literature from Nigeria. The knowledge of the prevalence of prevalence of pathogenic Leptospira serovars in these animals as a zoonotic risk to workers, zoo visitors and the general public is essential. This investigation was carried out on archival kidney and liver samples of captive and Zoological Garden animals (66) of an institutional facility, submitted for necropsy to the Department of Veterinary Pathology between the periods of 2010–2015. The gross diagnosis reports were obtained from the necropsy records, detection of pathogenic Leptospira serovars was by Warthin Starry silver staining and immunohistochemistry techniques using standard methods. Six samples out of the sixty-six samples were positive for leptospira four samples were positive by silver stain method, while two samples were positive by immunohistochemistry. In this study, serovar Pomona and grippotyphosa were detected in the foxes while serovar Pomona was detected in the horse. This study has revealed the presence of pathogenic leptospires in some captive wild and zoological garden animals.

Fluorescent Silver Staining of Proteins in Polyacrylamide Gels.

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The classic silver stains have certain drawbacks, such as high background staining, poor protein recovery, low reproducibility, a narrow linear dynamic range for quantification, and limited compatibility with mass spectrometry (MS). Now, with the use of a fluorogenic Ag+ probe, TPE-4TA, we developed a fluorescent silver staining method for the total protein visualization in polyacrylamide gels. This new stain avoids the troublesome silver reduction step in traditional silver stains. Moreover, the fluorescent silver stain demonstrates good reproducibility, sensitivity, and linear quantification in protein detection, making it a useful and practical protein gel stain.

Electron microscopy and tomography reveal that sodium 2-naphthalene sulfonate incorporated into perming solutions swells and tilts trichocyte intermediate filaments causing straightening of curly Japanese human hair.

A new hair-care process has been specifically developed for the straightening of curved Japanese woman's hair . The process included sodium 2-naphthalene sulfonate (SNS) in the reduction and oxidation steps of a conventional perming process. Our objective was to develop an understanding of how this process caused hair straightening by measuring the changes to morphology and ultrastructure between untreated, conventionally permed and SNS permed hair. Untreated and SNS permed Merino wool fibres were used to confirm structural changes. Japanese hair samples were measured for single-fibre curvature before and after perming treatments. A silver staining method was developed to stain hair fibres without changing fibre curvature so that transmission electron microscopy could be used to measure changes in the lateral dimensions of all structural components from the cellular to protein filament level. Electron tomography determined intermediate filament slopes and slope changes after SNS perming relative to the central longitudinal axis of the fibre. SNS perming was found to cause greater lateral swelling than conventional perming of: the paracortical cells of wool; the cuticle, the cuticular cell membrane complex and the macrofibrillar centre-to-centre distance of hair; and of the intermediate filaments in wool and hair. In curved hair, SNS perming caused the intermediate filaments of the helical macrofibrils to simultaneously swell and to tilt further, resulting in the slight longitudinal contraction of the macrofibrils. The overall swelling and tilting was greatest in the helical macrofibrils of Type B cortical cells predominately located in the convex fibre half. The presence of a higher percentage of helical macrofibrils in the convex fibre half than in the concave fibre half caused a contraction differential between the two halves leading to straighten of the curved fibre. A mechanical model was proposed to explain how SNS perming straightened curly hair. The effects of conventional and SNS perming on the morphological and ultrastructural components of curved Japanese hair and high-curl Merino wool fibres have given clear insights into understanding the mechanism of fibre curvature change.

Ribosomal DNA transcription in prefrontal pyramidal neurons is decreased in suicide

Prefrontal cortical regions, which are crucial for the regulation of emotionally influenced behaviour, play most probably a dominant role in the pathogenesis of suicide. The study was carried out on paraffin-embedded brain tissue blocks containing specimens from the anterior cingulate cortex (dorsal and ventral parts), the orbitofrontal cortex, and the dorsolateral cortex obtained from 23 suicide completers (predominantly violent) with unknown psychiatric diagnosis and 25 non-suicidal controls. The transcriptional activity of ribosomal DNA (rDNA) as a surrogate marker of protein biosynthesis was evaluated separately in layers III and V pyramidal neurons in regions of interest (ROIs) mentioned above by the AgNOR silver staining method bilaterally. The overall statistical analysis revealed a decrease of AgNOR area suggestive of attenuated rDNA activity in suicide victims versus controls, particularly in male subjects. Further ROI-specific post-hoc analyses revealed decreases of the median AgNOR area in suicides compared to non-suicides in all 16 ROIs. However, this effect was only significant in the layer V pyramidal neurons of the right ventral anterior cingulate cortex. Our findings suggest that decreased rDNA transcription in prefrontal pyramidal neurons plays possibly an important role in suicide pathogenesis.

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors.

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cellsand provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Redescription of a Hymenostome Ciliate, Tetrahymena setosa (Protozoa, Ciliophora) Notes on its Molecular Phylogeny

In recent years, Tetrahymena species have been used as model organisms for research in a wide range of fields, highlighting the need for a fuller understanding of the taxonomy of this group. It is in this context that this paper uses living observation and silver staining methods to investigate the morphology and infraciliature of one Tetrahymena species, T.setosa (Schewiakoff 1892 Verh. Naturh. Med. Ver. Heidelb., 4:544) McCoy (1975) Acta Protozool., 14:253; the senior subjective synonym of T.setifera Holz and Corliss (1956) J. Protozool., 3:112; isolated from a freshwater pond in Harbin, north-eastern China. This organism can be distinguished from other described Tetrahymena species mainly by its single caudal cilium, which is about twice the length of the somatic ciliature. While the Harbin isolate appears similar to the population described by Holz and Corliss (1956) J. Protozool., 3:112, an improved diagnosis for T.setosa is given based on the previous descriptions and the Harbin population. In summary, this species can be recognized mainly by the combination of the following characters: body invivo approximately 40μm × 25μm, 21-26 somatic kineties, one to four contractile vacuole pores associated with meridians 6-11 and a single caudal cilium. The small subunit ribosomal (SSU) rRNA gene and the cox1 gene sequences of Harbin population are also characterized in order to corroborate that the isolated species branches in phylogenetic trees as a T.setosa species. The phylogenetic analysis also indicated that sequences of populations of Tetrahymena species should be published with detailed morphological identifications.

Detection of Borrelia in Ixodes scapularis ticks by silver stain, immunohistochemical and direct immunofluorescent methods.

Lyme disease (LD) is one of the most common tick-borne diseases caused by several Borrelia species of spirochetes. Ixodes scapularis is responsible for the transmission of LD in the northeastern United States. The rate of infection varies with the duration of tick attachment to the host; however, this information may be unknown. In skin biopsies, it is often difficult to identify spirochetes. Testing of ticks is not routinely performed. Treatment is established by clinical presentation. To test paraffin-embedded I. scapularis ticks for Borrelia by different methods. We examined 20 paraffin-embedded ticks by silver stain, immunohistochemical (IHC) and direct immunofluorescent (DIF) methods and compared the percentage of positivity with DIF results from the Connecticut Agricultural Experiment Station. The results were similar by DIF, which proved to be the most sensitive method, followed by the silver stain and IHC. We found that the identification of spirochetes in paraffin-embedded ticks was less difficult than in tissue with a comparable turnaround time to that of a routine biopsy. Timely identification of Borrelia in ticks may influence the clinical management of the patients.

A Fast Silver Staining Protocol Enabling Simple and Efficient Detection of SSR Markers using a Non-denaturing Polyacrylamide Gel.

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver staining is a widely used method for the detection of SSR markers in a polyacrylamide gel. However, conventional protocols for silver staining are technically demanding and time-consuming. Like many other biological laboratory techniques, silver staining protocols have been steadily optimized to improve detection efficiency. Here, we report a simplified silver staining method that significantly reduces reagent costs and enhances the detection resolution and picture clarity. The new method requires two major steps (impregnation and development) and three reagents (silver nitrate, sodium hydroxide, and formaldehyde), and only 7 min of processing for a non-denaturing polyacrylamide gel. Compared to previously reported protocols, this new method is easier, quicker and uses fewer chemical reagents for SSR detection. Therefore, this simple, low-cost, and effective silver staining protocol will benefit genetic mapping and marker-assisted breeding by a quick generation of SSR marker data.

Fluorogenic Ag+ -Tetrazolate Aggregation Enables Efficient Fluorescent Biological Silver Staining.

Silver staining, which exploits the special bioaffinity and the chromogenic reduction of silver ions, is an indispensable visualization method in biology. It is a most popular method for in‐gel protein detection. However, it is limited by run‐to‐run variability, background staining, inability for protein quantification, and limited compatibility with mass spectroscopic (MS) analysis; limitations that are largely attributed to the tricky chromogenic visualization. Herein, we reported a novel water‐soluble fluorogenic Ag+ probe, the sensing mechanism of which is based on an aggregation‐induced emission (AIE) process driven by tetrazolate‐Ag+ interactions. The fluorogenic sensing can substitute the chromogenic reaction, leading to a new fluorescence silver staining method. This new staining method offers sensitive detection of total proteins in polyacrylamide gels with a broad linear dynamic range and robust operations that rival the silver nitrate stain and the best fluorescent stains.