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MethodsX 2021We designed a marking stand for the Dermojet , which substantially improves fish marking via needleless subcutaneous injection of dye. The marking stand allows to...
We designed a marking stand for the Dermojet , which substantially improves fish marking via needleless subcutaneous injection of dye. The marking stand allows to increase the nozzle-to-fish distance, adjust this position and to keep the jet injector fixed during operation as well as dye refilling. A laser pointer enables a precise and small-scale aiming. Using this marking stand we marked the caudal fin of small fish with Alcian blue for a flume experiment. In total we marked 204 gudgeon () and spirlin () of 9-14 cm length with up to two dots per fish. Weighing, measuring and marking one sedated fish took 30 to 60 s. Immediate marking success was 100%. Fish were kept indoors in tanks for 7-12 days post-marking and the colour mark remained visible for the complete study period. During our flume experiment the colour marks at the caudal fin were detectable on all fish regardless of swimming position. With this easy and fast method fish can be marked gently, reliably and efficiently.•Application of a high-pressure jet injector for needleless and accurate colour marking of fish.•Manual for marking the caudal fin of small fish with Alcian blue.
PubMed: 34754781
DOI: 10.1016/j.mex.2021.101510 -
Animals : An Open Access Journal From... May 2023In terrestrial mammals, the parotid and mandibular glands secrete different types of saliva into the oral cavity. Both glands were obtained from two female lowland...
In terrestrial mammals, the parotid and mandibular glands secrete different types of saliva into the oral cavity. Both glands were obtained from two female lowland tapirs () and one female aardvark () from the Wroclaw Zoological Garden (Poland) and examined by light microscopy (hematoxylin and eosin, mucicarmine, periodic acid-Schiff, Alcian blue pH 1.0, Alcian blue pH 2.5, Alcian blue pH 2.5/PAS, and Hale's dialysed iron). Both the parotid glands observed in the lowland tapir and aardvark were compound alveolar serous secretory units, and in both species, the secretion was composed of neutral and acidic mucopolysaccharides (sialo and sulfated mucins). However, in both the lowland tapir and aardvark, a histological examination found the stroma of the mandibular gland was divided into very large lobes by poorly marked connective tissue. While many interlobar and striated ducts were found in the aardvark, very few were found in the lowland tapir. The mandibular gland was a branched tubular (mucous secretion) type in the lowland tapir, but it was a branched tubuloalveolar (mucous-serous) type in the aardvark. In all tested glands, the secretion was composed of neutral mucopolysaccharides, acid-sulfated mucosubstances, and sialomucins.
PubMed: 37238113
DOI: 10.3390/ani13101684 -
Journal of Cytology 2024In endometrial cytology, differentiating endometrial glandular stromal breakdown (EGBD) from endometrial endometrioid carcinoma (G1-EEC) is often difficult. In this...
Staining Pattern of Alcian Blue in Endometrial Cytology: Utility in Distinguishing Grade 1-Endometrial Endometrioid Carcinoma from Endometrial Glandular Stromal Breakdown.
BACKGROUND AND OBJECTIVE
In endometrial cytology, differentiating endometrial glandular stromal breakdown (EGBD) from endometrial endometrioid carcinoma (G1-EEC) is often difficult. In this study, we provided a new focus on chondroitin sulfate (CS), a major substrate component of the endometrial stroma, and assessed the diagnostic utility of Alcian Blue (AB) staining in the differential diagnosis in liquid-based cytological (LBC) samples.
MATERIALS AND METHODS
LBC specimens from 19 patients with a proliferative endometrium, 36 with EGBD, and 30 with G1-EEC who underwent endometrial cytology were stained with AB (pH 1.0), and their reactivity was observed. In addition, immunocytochemical staining of CS and CD31 was performed for five cases each to evaluate their interrelationship with blood vessels.
RESULTS
Regarding the 30 G1-EEC cases, at least one of the three representative staining patterns was observed by AB staining: dot-like, microtubular, and finely branched linear patterns. Moreover, the inner portion of the tubular material observed by AB staining expressed CD31. Conversely, in the 36 EGBD cases, only five metaplastic clusters with irregular protrusions and condensed stromal clusters (CSCs) showed a dot-like positive pattern, and background CSCs did not show reactivity to AB staining in any of the cases. Furthermore, the vascular structure expressing CD31 in cell clusters was also unclear.
CONCLUSIONS
We demonstrated that AB staining shows different staining patterns in G1-EEC and EGBD, reflecting their different tissue structures. Our data provide new insights into endometrial cell diagnosis changes and demonstrate that AB staining is a potential new diagnostic aid tool for the differentiation of G1-EEC from EGBD.
PubMed: 38779603
DOI: 10.4103/joc.joc_121_23 -
Veterinary Pathology Mar 2009Utilization of a combined Alcian Blue and Pyronine Y histochemical method for the assessment of multiple parameters in the respiratory tract of various species is...
Utilization of a combined Alcian Blue and Pyronine Y histochemical method for the assessment of multiple parameters in the respiratory tract of various species is described. Acidic mucins were deep blue (sialylated mucins), red (sulfated mucins), or variably purple (mixture of sialylated/sulfated mucins), and differential mucus production was readily detected in a murine respiratory syncytial virus vaccine model of pulmonary inflammation. Elastic fibers stained red in the walls of pulmonary arteries, connecting airways, alveolar septa, and subpleural interstitium. Mast cells had red to red-purple granular cytoplasmic staining. Nuclei were ubiquitously counterstained pale blue. Representative staining was detected in tissues from multiple species, including inbred mice, rats, ferrets, cats, dogs, sheep, and pigs. The fluorescent property of the stained tissues offers additional modalities with which to analyze tissue sections. This histochemical technique detects multiple critical parameters in routine paraffin sections of lung tissue, reduces the need for repeated serial sectioning and staining, and is cost-effective and simple to perform.
Topics: Alcian Blue; Animals; Carnivora; Disease Models, Animal; Lung Diseases; Mice; Pyronine; Rabbits; Rats; Respiratory System; Sheep; Staining and Labeling; Swine
PubMed: 19261646
DOI: 10.1354/vp.46-2-325 -
Journal of Functional Biomaterials Apr 2019Accurate determination of the amount of glycosaminoglycans (GAGs) in a complex mixture of extracellular matrix (ECM) is important for tissue morphogenesis and...
Accurate determination of the amount of glycosaminoglycans (GAGs) in a complex mixture of extracellular matrix (ECM) is important for tissue morphogenesis and homeostasis. The aim of the present study was to investigate an accurate, simple and sensitive alcian blue (AB) method for quantifying heparin in biological samples. A method for analyzing heparin was developed and parameters such as volume, precipitation time, solvent component, and solubility time were evaluated. The AB dye and heparin samples were allowed to react at 4 ℃ for 24 h. The heparin-AB complex was dissolved in 25 N NaOH and 2-Aminoethanol (1:24 /). The optical density of the solution was analyzed by UV-Vis spectrometry at 620 nm. The modified AB method was validated in accordance with U.S. Food and Drug Administration guidelines. The limit of detection was found to be 2.95 µg/mL. Intraday and interday precision ranged between 2.14-4.83% and 3.16-7.02% (n = 9), respectively. Overall recovery for three concentration levels varied between 97 ± 3.5%, confirming good accuracy. In addition, this study has discovered the interdisciplinary nature of protein detection using the AB method. The basis for this investigation was that the fibrous protein inhibits heparin-AB complex whereas globular protein does not. Further, we measured the content of sulfated GAGs (sGAGs; expressed as heparin equivalent) in the ECM of decellularized porcine liver. In conclusion, the AB method may be used for the quantitative analysis of heparin in ECM scaffolds for tissue engineering applications.
PubMed: 31052349
DOI: 10.3390/jfb10020019 -
American Journal of Translational... 2023The type X collagen gene () is a signature gene of hypertrophic chondrocytes that are known as the main engine of long bone growth. Multiple transcription factors (TFs),...
BACKGROUND
The type X collagen gene () is a signature gene of hypertrophic chondrocytes that are known as the main engine of long bone growth. Multiple transcription factors (TFs), including myocyte enhancer factor 2A (Mef2a), have previously been identified by analysis as potential gene regulators.
OBJECTIVES
In this study, we aimed to investigate the correlation between Mef2a and Col10a1 expression and the possible effects on chondrocyte proliferation and hypertrophic differentiation .
METHODS
First, Mef2a expression in proliferating and hypertrophic chondrocytes were detected by quantitative real-time PCR (qRT-PCR) and Western blotting in two chondrocytic models, ATDC5 and MCT cells, as well as in mouse chondrocytes . Transfection with Mef2a small interfering fragments or Mef2a overexpression plasmids in the above chondrocytic models were performed to determine how Mef2a knockdown or overexpression may influence Col10a1 expression. The binding between Mef2a and its putative binding site within the 150 bp cis-enhancer which was evaluated by the dual luciferase reporter assay. The effect of Mef2a on chondrocyte differentiation was determined by examining the chondrogenic marker gene expression by qRT-PCR and by alcian blue, alkaline phosphatase (ALP), and alizarin red staining of the ATDC5 cells stably knocked down by Mef2a.
RESULTS
The expression of Mef2a in hypertrophic chondrocytes was significantly higher than that in proliferative chondrocytes in both chondrocytic models as well as in mouse chondrocytes . Interference with Mef2a caused decreased Col10a1 expression, while overexpression of Mef2a upregulated Col10a1. The result of the dual luciferase reporter assay showed that Mef2a enhanced Col10a1 gene enhancer activity via its putative Mef2a binding site. For the staining of ATDC5 stable cell lines, although no significant differences were seen in ALP staining, significantly weaker alcian blue staining intensity was noticed in Mef2a knockdown stable cell lines compared to the control cells at day 21, while slightly weaker alizarin red staining was seen in the stable cell lines at days 14 and 21. Correspondingly, we detected decreased runt-related transcription factor 2 (), increased SRY-box transcription factor 9 (), as well as differential expression of other chondrogenic markers in ATDC5 stable cell lines compared with the controls.
CONCLUSIONS
In conclusion, our results support that Mef2a upregulates Col10a1 expression possibly by interaction with its cis-enhancer. Altered levels of Mef2a affects the expression of chondrogenic marker genes, such as Runx2 and Sox9, but may only play an insignificant role during chondrocyte proliferation and maturation.
PubMed: 37434855
DOI: No ID Found -
Heliyon Oct 2020The efficacy of mesnchymal stem cells (MSCs) to treat the necrotic tissue of salivary glands (SGs) has yet investigated.
BACKGROUND
The efficacy of mesnchymal stem cells (MSCs) to treat the necrotic tissue of salivary glands (SGs) has yet investigated.
OBJECTIVE
This study was conducted to investigate the potential capacity of MSCs to restore the function and regenerate the necrotic submandiular gland in the rat animal model.
METHODS
Twenty-one Sprague-Dawley rats were provided from a breeding colony and randomly divided into three groups including the positive control or induced SG atrophy without treatment, the treatment group or induced SG atrophy with MSCs isolated transplantation and the negative control group consists of healthy rats. The atrophic and necrotic submandiular gland was induced using intraoral duct ligation of the main duct of submandiular gland for one month. The isolated stem cells were confirmed using flow cytometry for CD90 and CD 105. The isolated MSCs were cultured and injected to submandiular gland and the potential efficacy of MSCs to treat the atrophic submandibular glands was evaluated using histopathology on two weeks post-transplantation. To detect the acinar cell protein secretory granules, Alcian Blue and periodic acid shift (PAS) staining were done. For the demonstration of mitotic index or proliferation rate of the SG epithelia tissue, Ki-67 and Smbg proteins expression were evaluated using immunohistochemistry.
RESULTS
The locally injected MSCs could regenerate the overall histological structure of the necrotic submandibular gland tissue within 2 weeks of post-transplantation. Alcian Blue and PAS staining indicated that the mean amount of serous and mucin secretions in the treatment group was significantly increased compared to the positive control groups. We have also found that the treatment group significantly express higher Ki-67 protein, as a diagnostic marker for cell mitosis and proliferation rate, and lower Smbg protein, as a diagnostic marker, for damage to the submandibular gland than that of control group.
CONCLUSION
This study demonstrates the therapeutic benefits of MSCs isolated from the SG in treating atrophic and necrotic SGs in a rat model. MSCs may be potential candidates for cell-based therapies targeting hypofunction of SG induced by a range of diseases or because of surgery and radiotherapy of head and neck cancers.
PubMed: 33083616
DOI: 10.1016/j.heliyon.2020.e05162 -
JOR Spine Mar 2024Intradiscal transplantation of mesenchymal stromal cells (MSCs) has emerged as a promising therapy for intervertebral disc degeneration (IDD). However, the hostile...
BACKGROUND
Intradiscal transplantation of mesenchymal stromal cells (MSCs) has emerged as a promising therapy for intervertebral disc degeneration (IDD). However, the hostile microenvironment of the intervertebral disc (IVD) may compromise the survival of implanted cells. Interestingly, studies reported that paracrine factors, such as extracellular vesicles (EVs) released by MSCs, may regenerate the IVD. The aim of this study was to investigate the therapeutic effects of Wharton's Jelly MSC (WJ-MSC)-derived EVs on human nucleus pulposus cells (hNPCs) using an in vitro 3D alginate-bead culture model.
METHODS
After EV isolation and characterization, hNPCs isolated from surgical specimens were encapsulated in alginate beads and treated with 10, 50, and 100 μg/mL WJ-MSC-EVs. Cell proliferation and viability were assessed by flow cytometry and live/dead staining. Nitrite and glycosaminoglycan (GAG) content was evaluated through Griess and 1,9-dimethylmethylene blue assays. hNPCs in alginate beads were paraffin-embedded and stained for histological analysis (hematoxylin-eosin and Alcian blue) to assess extracellular matrix (ECM) composition. Gene expression levels of catabolic (), anabolic (), and hNPC marker () genes were analyzed through qPCR. Collagen type I and type II content was assessed with Western blot analysis.
RESULTS
Treatment with WJ-MSC-EVs resulted in an increase in cell content and a decrease in cell death in degenerated hNPCs. Nitrite production was drastically reduced by EV treatment compared to the control. Furthermore, proteoglycan content was enhanced and confirmed by Alcian blue histological staining. EV stimulation attenuated ECM degradation and inflammation by suppressing catabolic and inflammatory gene expression levels. Additionally, NPC phenotypic marker genes were also maintained by the EV treatment.
CONCLUSIONS
WJ-MSC-derived EVs ameliorated hNPC growth and viability, and attenuated ECM degradation and oxidative stress, offering new opportunities for IVD regeneration as an attractive alternative strategy to cell therapy, which may be jeopardized by the harsh microenvironment of the IVD.
PubMed: 38222813
DOI: 10.1002/jsp2.1274 -
JAAD Case Reports May 2016
PubMed: 27408935
DOI: 10.1016/j.jdcr.2016.04.005 -
Anatomical Record (Hoboken, N.J. : 2007) May 2021Bone and cartilage staining has provided anatomists with the ability to generate detailed descriptions of the adult and developing skeleton. Typically, Alizarin red S...
Bone and cartilage staining has provided anatomists with the ability to generate detailed descriptions of the adult and developing skeleton. Typically, Alizarin red S and Alcian blue are used for the staining of bone and cartilage, respectively. The binding of Alizarin red S and calcium is most stable at basic conditions, however, Alcian blue exhibits specific binding to polyanionic substances such as mucopolysaccharides under acidic conditions. Typical bone and cartilage staining protocols are conducted under acidic conditions. Because of this discrepancy in optimal pH, issues can arise in the staining of small specimens such as larval fish. Specifically, staining embryonic or larval specimens under acidic conditions can cause decalcification of small bones. Decalcification can completely inhibit the uptake of Alizarin red S in small bones. In order to mitigate this issue, researchers have developed an acid-free staining protocol that utilizes the concept of critical electrolyte concentration. While many researchers have adopted acid-free bone and cartilage staining, some researchers continue to stain these small specimens with acidic staining protocols. To ensure the reliability and validity of our skeletal descriptions, we urge scientists to utilize acid-free staining protocols when analyzing the skeletons of larval or embryonic specimens.
Topics: Alcian Blue; Animals; Bone and Bones; Cartilage; Staining and Labeling
PubMed: 33026708
DOI: 10.1002/ar.24526