Rapid and Efficient Investigation of Ciliate Nuclear Development During Conjugation Through Optimizing Hoechst33342 Staining Workflow

Ciliates are eukaryotic unicellular organisms with complex morphology and developmental processes,including asexual and sexual processes.Conjugation is a form of sexual process that renews genetic materials.However,visualizing conjugation in ciliates is a challenge due to the complexity and dynamics of the process,while traditional staining methods are often insufficient for the research.This study introduces a new method for visualizing developmental progression in the nuclei during conjugation using Hoechst33342 staining.It describes how to proceed from cell culture,conjugation induction and synchronization,staining preparation,and observation to statistical analysis.The combination of fluorescent staining with the‘volume-fixing'technique eliminates the fixation and dehydration steps,thus reducing the overall operation time to just 20 minutes.This method offers several advantages over traditional staining techniques for studying the nuclei during conjugation.It improves image quality and workflow efficiency and enables real-time observation of live cell states.Potential solutions to challenges that may arise during experimental procedures are introduced and references and guidelines for cytological research are provided in this paper.

Guidelines for the standardized diagnosis and treatment of non-specific orbital inflammation(2024)

Non-specific orbital inflammation(NSOI)is a noninfectious orbital inflammation.Although it is often considered the most common diagnosis in orbital biopsies,it is an exclusionary diagnosis that requires ruling out systemic disease or other possible causes.Its characteristics include acute orbital signs and symptoms,including pain,proptosis,periorbital edema,chemosis,diplopia,and visual impairment.The clinical manifestations and histological findings of NSOI are heterogeneous,without specific diagnostic criteria or treatment guidelines,which poses significant challenges for diagnosis and treatment.This guideline provides a detailed description of the definition,classification,diagnosis,and treatment of NSOI.

A comparing study of quantitative staining techniques for retinal neovascularization in a mouse model of oxygen-induced retinopathy

AIM: To explore an efficient, practical and objective quantitative method to evaluate the retinal neovascularization in mouse model of oxygen induced retinopathy (OIR). METHODS: Thirty C57BL/6J mice were explored in OIR model procedure. Eyes were removed for different staining methods including: (1) HE staining; (2) immunohistochemistry with Griffonia Simplicifolia Lectin (GSL); (3) Immunofluorescence with FITC labeled CD31 antibody; (4) Two-step immunofluorescence with purified-CD31 antibody; (5) FITC-Dextran perfusion combined with two-step purified-CD31immunofluorescence. Images of the retinal vasculature were analyzed by imaging software. ' RESULTS: GSL immunohistochemistry could clearly demonstrate the deep and superficial capillary beds. FITC labeled CD31 Immunofluorescence was blurring with high fluorescence background which was hard to distinguish retinal neovascularization in some area. Excellent detail of neovascularization and preexistent retinal vessels was provided in two-step Purified-CD31 immunofluorescence group. CONCLUSION: GSL immunohistochemistry can clearly demonstrate neovascularization tufts in deep and superficial capillary beds. Immunofluorescence of specific antigen CD31 on vascular endothelium can selectively label the neovascularization of mouse retina. When combined with computer analysis software, it is an effective and objective quantitative method to evaluate the retinal neovascularization in OIR mouse model.

Role of MMP-2 and MMP-9 and their natural inhibitors in liver fibrosis, chronic pancreatitis and non-specific inflammatory bowel diseases

BACKGROUND： There is a growing evidence that matrix metalloproteinase （MMP）-2 and MMP-9 （gelatinases） play an important role in the pathogenesis of numerous disorders, especially with inflammatory etiology and extracellular matrix （ECM） remodeling. Despite the fact that gelatinases involve in liver cirrhosis is provided in the literature, their role in the pathogenesis of chronic pancreatitis and non-specific inflammatory bowel diseases is still under investigation. DATA SOURCES： We carried out a PubMed search of Englishlanguage articles relevant to the involvement of gelatinases in the pathogenesis of liver fibrosis, pancreatitis, and non-specific inflammatory bowel diseases. RESULTS： The decreased activity of gelatinases, especially MMP-2, is related to the development of liver fibrosis, probably due to the decrease of capability for ECM remodeling. Similar situation can be found in chronic pancreatitis; however, reports on this matter are rare. The presence of non-specific inflammatory bowel diseases results in MMP-9 activity elevation. CONCLUSION： The fluctuation of gelatinases activity during liver fibrosis, chronic pancreatitis and non-specific inflammatory bowel diseases is observed, but the exact role of these enzymes demands further studies.

Influences of immersion bathing in Bacillus velezensis DY-6 on growth performance,non-specific immune enzyme activities and gut microbiota of Apostichopus japonicus

In this study, the influences of immersion bathing in different concentrations of Bacillus velezensis DY-6 on the body weight gain rate and non-specific immune enzyme activities of the coelom fluid of sea cucumber (Apostichopus japonicus) were determined in order to obtain the optimum bacterial concentration. The gut microbiota change in A. japonicus was then analyzed through high-throughput sequencing during the immersion bathing in B. velezensis DY-6 at the optimum concentration for 49 d. The results illustrate that the body weight growth rate of all bathing groups was higher than that of the control. The highest growth rate (25.3%) was achieved when the bacterial concentration was 1×10^3 CFU/mL. The activities of non-specific immune enzymes (ACP, AKP, SOD and LZM) of all bathing groups increased, and the activities of the enzymes of groups bathed with the bacterium at 1×10^3 and 1×10^4 CFU/mL reached the highest on day 21 and day 28. Taking the growth rate and economic cost into consideration, the optimum concentration of B. velezensis DY-6 was 1×10^3 CFU/mL. The influences of immersion bathing in B. velezensis DY-6 at 1×10^3 CFU/mL on the gut microbiota of A. japonicus were then evaluated through 16S rDNA sequencing analysis. Results showed that the gut microbiota changed with the addition of B. velezensis DY-6, and the richness and diversity of the gut microbiota peaked twice on day 14 and day 21, respectively. In association with the non-specific immune enzyme activities and if day 28 was selected as the dividing point, the community structure of the gut microbiota could be obviously divided into two types. The correlation analysis revealed that the non-specific immune enzyme activities were correlated significantly to some gut bacteria (in the phyla Firmicutes, Proteobacteria, and Bacteroidetes) after immersion bathing in B. velezensis DY-6. Our findings will provide the theoretical foundation for probiotic application in sea cucumber farming.

Mouse Karyotype Obtained by Combining DAPI Staining with Image Analysis

In this study, mitotic metaphase chromosomes in mouse were identified by a new chromosome fluorescence banding technique combining DAPI staining with image analysis. Clear 4＇, 6-diamidino-2-phenylindole （DAPI） multiple bands like （J-hands could be produced in mouse. The Meta- Morph software was then used to generate linescans of pixel intensity for the banded chromosomes from short arm to long arm. These linescans were sufficient not only to identify each individual chromosome but also analyze the physical sites of bands in chromosome. Based on the results, the clear and accurate karyotype of mouse metaphase chromosomes was established. The technique is therefore considered to he a new method for cytological studies of mouse.

Selection of oocytes for in vitro maturation by brilliant cresyl blue staining： a study using the mouse model

Selecting oocytes that are most likely to develop is crucial for in vitro fertilization and animal cloning. Brilliant cresyl blue （BCB） staining has been used for oocyte selection in large animals, but its wider utility needs further evaluation. Mouse oocytes were divided into those stained （BCB＋） and those unstained （BCB-） according to their ooplasm BCB coloration. Chromatin configurations, cumulus cell apoptosis, cytoplasmic maturity and developmental competence were compared between the BCB＋ and BCB- oocytes. The effects of oocyte diameter, sexual maturity and gonadotropin stimulation on the competence of BCB＋ oocytes were also analyzed. In the large- and medium-size groups, BCB＋ oocytes were larger and showed more surrounded nucleoli （SN） chromatin configurations and higher frequencies of early atresia, and they also gained better cytoplasmic maturity （determined as the intracellular GSH level and pattern of mitochondrial distribution） and higher developmental potential after in vitro maturation （IVM） than the BCB-oocytes. Adult mice produced more BCB＋ oocytes with higher competence than the prepubertal mice when not primed with PMSG. PMSG priming increased both proportion and developmental potency of BCB＋ oocytes. The BCB＋ oocytes in the large-size group showed more SN chromatin configurations, better cytoplasmic maturity and higher developmental potential than their counterparts in the medium-size group. It is concluded that BCB staining can be used as an efficient method for oocyte selection, but that the competence of the BCB＋ oocytes may vary with oocyte diameter, animal sexual maturity and gonadotropin stimulation. Taken together, the series of criteria described here would allow for better choices in selecting oocytes for better development.

Effect of Dietary Alanyl-glutamine Supplementation on Growth Performance, Development of Intestinal Tract, Antioxidant Status and Plasma Non-specific Immunity of Young Mirror Carp(Cyprinus carpio L.)

Exogenous alanyl-glutamine（Aln-Gln） was evaluated for its effects on growth performance, intestinal structure and function, antioxidant status and non-specific immunity of young carp（Cyprinus carpio L.）. Six diets supplemented with 0, 2.5, 5.0, 7.5, 10.0, or 15.0 g · kg-1 of Aln-Gln were fed to fish for 12 weeks. Supplementation with 7.5, 10.0, or 15.0 g · kg-1 of Aln-Gln significantly increased weight gain rate（WGR）, protein efficiency ratio（PER）, but feed conservation rate（FCR） and survival were not affected（P〉0.05）. The intestinal fold height and number, digestive enzyme, Na＋, K＋-ATPase activities was found to be significantly high（P〈0.05） with increasing dietary Aln-Gln supplementation up to 7.5 g · kg-1, but there were no significant differences for Aln-Gln supplementation from 7.5 to 15.0 g · kg-1. The glutathione peroxidase（GPX） activity, glutathione（GSH）, superoxide dismutase（SOD） activity increased and malondialdehyde（MDA） levels decreased significantly（P〈0.05） in the intestine, hepatopancreas, plasma and muscles. The plasma complement-3（C3） and complement-4（C4） levels were significantly（P〈0.05） improved at 5.0 g · kg-1 level and decreased when over 7.5 g · kg-1. The plasma lysozyme（LSZ） activity increased significantly（P〈0.05） at 7.5, 10.0, or 15.0 g · kg-1 level. In summary, the results showed that Aln-Gln improved growth performance, development and function of the intestine, the activity of the antioxidant defense system and the plasma non-specific immunity of the carps. The optimal Aln-Gln level was 8.24 g · kg-1 diet for WGR based on broken-line regression model analysis.

Application of an indirect immunofluorescent staining method for detection of Salmonella enteritidis in paraffin slices and antigen location in infected duck tissues

AIM： To detect Salmonella enteritidis （S. enteritidis） in paraffin slices and antigen location in infected duck tissues. METHODS： The rabbits were immunized with purified bacillus to obtain S. enteritidis-specific antibody, which were then extracted by the caprylic-ammonium sulphate method, purified through High-Q columns. An indirect immuno-fluorescent staining method （IFA） was established to detect the S. enteritidis antigen in paraffin slices. Detected S. enteritidis in each organ tissue of ducklings experimentally infected with S. enteritidis. RESULTS： The gland of Garder, heart, kidney, spleen, liver, brain, ileum, jejunum, bursa of Fabricius from S. enteritidis experimentally infected ducklings were positive or strongly positive, and the S. enteritidis antigen mainly distributed in the infected cell cytoplasm.CONCLUSION： IFA is an intuitionist, sensitive and specific method in detecting S. enteritidis antigen in paraffin wax slices, and it is a good method in diagnosis and antigen location of S. enteritidis. We also conclude that the gland of Garder, heart, kidney, spleen, liver, ileum, jejunum are target organs in S. enteritidis infections of duck, and S. enteritidis is an intracellular parasitic bacterium.

Carbohydrate malabsorption in patients with non-specific abdominal complaints

Non-specific abdominal complaints are a considerable problem worldwide. Many patients are affected and many differential diagnoses have to be considered. Among these, carbohydrate malabsorption seems to play an important role. However, so far, only incomplete absorption of lactose is broadly accepted, while the malabsorption of fructose and sorbitol is still underestimated, although in many parts of the world it is much more frequent. Despite the success of dietary interventions in many patients, there are still a lot of unanswered questions that make further investigations

Comparison ofβ-Amyloid Plaque Labeling Methods:Antibody Staining,Gallyas Silver Staining,and Thioflavin-S Staining

Objective To evaluate senile plaque formation and compare the sensitivity of three differentβ-amyloid(Aβ)labeling methods(antibody staining,Gallyas silver staining,and thioflavin-S staining)to detect Aβdeposition.Methods APPswe/PSEN1dE9 transgenic mice(APP/PS1)of different ages were used to examine spatiotemporal changes in Aβplaque deposition.Antibody staining,Gallyas silver staining,and thioflavin-S staining were used to detect Aβplaque deposition in the same brain region of adjacent slices from model mice,and the results were compared.Results With aging,Aβplaques first appeared in the cortex and then the deposition increased throughout the whole brain.Significantly greater plaque deposition was detected by 6E10 antibody than that analyzed with Gallyas silver staining or thioflavin-S staining(P<0.05).Plaque deposition did not show significant difference between the APP/PS1 mice brains assayed with Gallyas silver staining and ones with thioflavin-S staining(P=0.0033).Conclusions The APP/PS1 mouse model of Alzheimer’s disease could mimick the progress of Aβplaques occurred in patients with Alzheimer’s disease.Antibody detection of Aβdeposition may be more sensitive than chemical staining methods.

Application of Prussian blue staining in the diagnosis of ocular siderosis

AIM:To explore the value of Prussian blue staining in the diagnosis of ocular siderosis.METHODS:Between January 2012 and January 2013,the Prussian blue stain used in anterior lens capsule and vitreous liquid after centrifugation from patients with definitive diagnosis and suspicious diagnosed of ocular siderosis. At the same time, give a negative control.RESULTS:Anterior lens capsule membrane and liquid of vitreous cavity from patients with definitive diagnosis and suspicious diagnosed of ocular siderosis revealed ferric ions that stained positively with Prussian blue. In the control group, there is no positive reaction.CONCLUSION:Prussian blue staining in the diagnosis of ocular siderosis has a very significant worth,suspected cases can be definitive diagnosed.

Effects of Size Grading on Growth and Non-Specific Immunity Factors of the Shrimp Litopenaeus vannamei Boone

The study aims to determine the effects of graded farming on growth performance and non-specific immunity factors of shrimp Litopenaeus vannamei Boone. Three size groups of shrimp, i.e., the small size group [Gs, with an average body length （BL） of （3.04 ± 0.36） cm and body weight （BW） （0.412± 0.35） g], the large group [GL, with a BL of （4.29±0.55） cm and BW of （1.098 ±0.42） g], and the ungraded group [Gm, with a BL of （3.47±0.81） cm and BW of （0.611 ±0.79） g], were reared under the same conditions for 8 wk. Growth performance and non- specific immunity factors were measured. The results showed that BW gain, biomass gain and the specific growth rate of body length （SGRL） were significantly influenced by size grading （one-way ANOVA, P 〈 0.05）. The peroxidase （POD） and antibacterial （Ua） activities of GL were lower than those of G. Superoxide dismutase （SOD） and lysozyme （U1） activities of Gm were lower than those of G. No significant difference （P = 0.121 〉 0.05） was found on phenoloxidase （PO） activity among the three size groups. Synthetically, size grading could enhance growth and rearing efficiency, and did not have a significant influence on the immunity of L. vannamei Boone. Therefore, graded fanning in L. vannamei Boone was feasible in the culture practice.

Preparation, optical properties and cell staining of water soluble amine-terminated PAMAM G2.0-Au nanocomposites

The solution chemical and optical characteristics of formation of amine-terminated polyamidoamine dendrimer G2.0（NH2-PAMAM G2.0）-Au nanocomposites in the aqueous solution of NH2-PAMAM G2.0 at various mole ratios of Au（Ⅲ） to NH2-PAMAM G2.0 were studied by both UV-visible spectrometry and fluorospectrometry. The NH2-PAMAM G2.0-Au nanocomposites, with a type of structure in which one Au nanoparticle is surrounded by several NH2-PAMAM G2.0 dendrimers, emit strong bluish violet fluorescence, and are uniform, water soluble and biocompatible as well as very stable in frozen conditions. The size of gold nanoparticles in the nanocomposites is about 2.5 nm and decreases with the increase of NH2-PAMAM G2.0 concentration. The NH2-PAMAM G2.0 plays an important role in acting as host or micro-reactor for Au（Ⅲ） before Au（Ⅲ） reduction and acting as dispersant and stabilizer for gold nanoparticles after Au（Ⅲ） reduction. Preliminary experiments of cells staining to human embryonic lung fibroblast cell lines show that the NH2-PAMAM G2.0-Au nanocomposites can be used as optical imaging markers for bioanalyses and medical diagnoses.

Screening of some indigenous herbal dyes for use in plant histological staining

The efficacies of some indigenous herbal dyes for use in staining plant materials were examined to obtain non-toxic, eco-friendly and cheap stains for use in plant histology. Dye extracts from Bixa orellana, Curcuma domestica, Lonchocarpus cyanescens and Pterocarpus osun were used to stain wood sections using the existing standard staining procedures with little modification. All the extracts had affinity for the fibre and vessel elements except the extract from L. cyanescens. The extracts from C. domestica and B. orellana had higher selectivity than those ofP. osun for fibre. From the results of the absorbance curves, each of the dye extracts from all speciese had minimum of two peaks, indicating that they had two or more colour imparting chromophores except dye extract from C. domestica. All the dye extracts were acidic with pH range of 3.77 to 6.77. Therefore, this study shows that dye extracts from B. orellana, C. domestica and P. osun could be solitarily or in combination with artificial dyes for plant histological staining.

Pollen viability of Polygala paniculata L.(Polygalaceae)using different staining methods

Polygala paniculata L.is a medicinal plant that grows in the Brazilian Atlantic coast,known as‘barba-de-São-João’,‘barba-de-bode’,‘vassourinha branca’,and‘mimosa’.In this study,pollen viability was estimated by three different staining methods:2%acetic orcein,2%acetic carmine,and Alexander’s stain.The young inflorescences of twenty accessions were collected and fixed in a solution of ethanol:acetic acid(3:1)for 24 hours,then stored in ethanol 70%under refrigeration.Six slides per plant,two for each stain,were prepared by squashing,and 300 pollen grains per slide were analyzed.Pollen viability was high(>70%)for most accessions of P.paniculata using the Alexander’s stain,which proved the most adequate method to estimate pollen viability.

Evaluation of sperm mitochondrial function using rh123/PI dual fluorescent staining in asthenospermia and oligoasthenozoospermia

Objective：The recent advent of flow cytometry（FCM）,coupled with fluorescent dyes,has been successfully applied to assess mitochondrial function.The aim of this study was to investigate the feasibility and clinical significance of detecting sperm mitochondrial function and to evaluate sperm mitochondrial function by using Rhodamine 123/propidium（Rh123/PI）dual fluorescent staining and FCM in asthenospermia and oligoasthenozoospermia.Methods：Twenty-five fertile men（with normal sperm parameters）and 230 infertile patients were examined.Fifty-five patients of the above 230 patients were selected for idiopathic infertility samples and were divided into two groups：asthenospermia（n=30）and oligoasthenozoospermia（n=25）.Rh123/PI dual fluorescent staining and FCM were carried out to examine sperm mitochondrial function.Results：Significant differences were found between the normal and abnormal semen samples（P0.05）when Rh123＋/PI-,Rh123-/PI＋and Rh123-/PI-sperm were examined by FCM,but there was no significant difference between the asthenospermia（P=0.469） and oligoasthenozoospermia group（P=0.950）when Rh123＋/PI-and Rh123-/PI＋sperm were then examined;however,a significant difference was found between the 2 groups（P=0.003）when Rh123-/PI-sperm were examined.There was no correlation between Rh123-/PI-sperm and semen parameters in the normal group,but there was a significant negative correlation between the sperm concentration and Rh123-/PI-sperm in asthenospermia and oligoasthenozoospermia patients（r=-0.509,-0.660;P=0.018,0.038）.Conclusion：Rh123/PI dual fluorescent staining and FCM can provide reliable information to assess the quality of sperm and reveal differences in mitochondrial membrane potential in asthenospermia and oligoasthenozoospermia.

Broadband seismic illumination and resolution analyses based on staining algorithm

Seismic migration moves reflections to their true subsurface positions and yields seismic images of subsurface areas. However, due to limited acquisition aperture, complex overburden structure and target dipping angle, the migration often generates a distorted image of the actual subsurface structure. Seismic illumination and resolution analyses provide a quantitative description of how the above-mentioned factors distort the image. The point spread function （PSF） gives the resolution of the depth image and carries full information about the factors affecting the quality of the image. The staining algorithm establishes a correspondence between a certain structure and its relevant wavefield and reflected data. In this paper, we use the staining algorithm to calculate the PSFs, then use these PSFs for extracting the acquisition dip response and correcting the original depth image by deconvolution. We present relevant results of the SEG salt model. The staining algorithm provides an efficient tool for calculating the PSF and for conducting broadband seismic illumination and resolution analyses.

Fluorescent vital staining of plant sexual cell nuclei with DNA-specific fluorochromes and its application in gametoplast fusion

DNA-binding fluorochromes are often used for vital staining of plant cell nuclei. However, it is not always sure whether the cells after staining still remain in living state. We chose several criteria to estimate the validity of real vital staining for sexual cell nuclei. These were: the cytoplasmic streaming in pollen tubes whose nuclei were stained, the simultaneous visualization of fluo-rochromatic reaction and nucleus staining in isolated generative cells, and the capability of isolated, prestained generative or sperm cells to fuse with other protoplasts. The results confirmed that 4',6-diamidino-2-phenylindole (DAPI), Hoechst 33258 and mithramycin could be used as real vital stains, though their efficiency varied from case to case; among them DAPI showed best effect. The fluorescent vital staining technique offered a useful means fori-dentification and selection of heterokaryons in gametoplast manipulation studies.

Effects of the Chinese Medicinal Herb Com plex Additives on Non-specific Immunity of Amursturgeon( Acipenser schrencki Brandt)

This study was to investigate the effects of aqueous decotion from three Chinese medicinal herb additives （ Cyrtomium fortunei, prescription I and prescription II） on non-specific immunity of 1 ＋ age old Amur sturgeon （Acipenser schrencki Brandt） by oral perfusion. Cyrtomiumfortunei, prescription I and prescription II were orally given to 15 fishes for each experimental group once a day, with the concentration of 9,175 and 36 g/S0 kg body weight, respectively. The administration was lasted for 14 days. Meanwhile, distilled water was orally given as the control. After the experiment, some of the experimental fishes were stimulated with high temperature （30 ℃ ） for two hours. Sampling was performed from fishes treated at 22 ℃ and 30 ℃ for measuring the protein content in the serum, phagocytic activity of leucocytes and the activity of the lysozyme in six tissues. The results indicated that Cyrtomiumfortunei and prescription II could help to enhance the content of various proteins and the phagocytic activity of leucocytes. There was no significant change in the effects between these two additives at either the normal temperature or high temperature. Prescription I did not show an obvious effect on the immunity of fishes at the normal temperature, but it did in promo- ting high-efficiency response and improving immunoregulation of fish shocked by some stimuli.