Mayer's Hematoxylin Stain Research Articles

Inhibitory effect of ginsenglactone A from Panax ginseng on the tube formation of human umbilical vein endothelial cells and migration of human ovarian cancer cells

BackgroundHere, we aimed to assess the inhibitory effect of a new compound from Panax ginseng on the migration of human ovarian cancer cells and tube formation of human umbilical vein endothelial cells (HUVECs). MethodsA new compound, ginsenglactone A (1), was isolated from ginseng roots, together with seven known compounds (2-8). Spectroscopic data were used to elucidate the chemical structure of 1. The tubular structure formation in HUVECs was assessed by Mayer's hematoxylin staining. The migration of A2780 cells was evaluated using the scratch wound healing assay. ResultsHUVECs treated with 1 had the statistically significant decrease in tubular structure formation compared to the HUVECs treated with compounds 2-8. This effect was enhanced by co-treatment with inhibitors for phosphatidylinositol 3-kinase (PI3K) (LY294002) and extracellular signal-regulated kinase (ERK) (U0126). Treatment with 1 decreased the expression of phosphorylation of ERK, PI3K, vascular endothelial growth factor receptor2 (VEGFR2), Akt, and mammalian target of rapamycin (mTOR). In addition, the ability of A2780 cells to cover the scratched area were also decreased. This effect was enhanced by co-treatment with U0126. Lastly, treatment with 1 decreased the phosphorylation of ERK, matrix metalloproteinase-9 (MMP-9), and MMP-2. ConclusionThese results suggest that ginsenglactone A is a potential inhibitor of HUVEC tubular structure formation and A2780 cellular migration, which may be helpful for understanding its anticancer mechanism.

Characterization and hepatoprotective evaluation of sesquiterpenes and diterpenes from the aerial parts of Juniperus sabina L.

Previously, we reported on the hepatoprotective activity of the total extract of Juniperus sabina L. against CCl4 induced liver toxicity in experimental animals. Biologically directed phytochemical study was conducted to identify the active compounds. Male Wistar rats and the standard drug silymarin were used in the study. Hepatoprotective activity was evaluated via serum biochemical parameters such as aspartate amino transferase (AST), alanine aminotransferase (ALT), gamma glutamyl transpeptidase (GGT), alkaline phosphatase (ALP) and total bilirubin. Tissue parameters including non-protein sulfhydryl groups (NP-SH), malonaldehyde (MDA) and total protein (TP) were also determined. Histopathological study was conducted utilizing Mayer’s hematoxylin stain, Periodic Acid Schiff – Hematoxylin (PAS-H) and Masson trichrome technique on light microscope. Electron microscope images were also generated for the study. The activity of the total extract was trapped to the petroleum ether fraction after liquid-liquid fractionation where 51% reduction in the levels of AST, bilirubin and 44% in the levels of ALT were observed. Chromatographic purification of the petroleum ether fraction resulted in the isolation of nine compounds namely: trans-calamenene (1), cadalene (cadalin) (2), epi-cubenol (3), manool (4), calamenene-10β-ol (5), calamenene-10α-ol (6), 4-epi-abietic acid (7), sandaracopimaric acid (8) and isopimaric acid (9). Compounds 1–3, 5 and 6 are belonging to cadinane sesquiterepenes, while compounds 4, 7–9 were of diterpene skeleton. The major compounds were tested for their hepatoprotective effect. Compounds 3 showed marked improvement in the levels of AST and ALT, compound 4 was effective in improving the levels of AST, ALT, GGT, ALP and bilirubin, while compound 7 showed significant improvement in GGT, ALP and bilirubin levels.

Inhibiting wear particles-induced osteolysis with naringin

The purpose of this study was to determine the effects of naringin on osteoclastogenesis and osteolysis both in vitro and in vivo. In this research osteoclasts were generated from mouse bone marrow monocytes with the receptor activator of NF-КB ligand and the macrophage colony stimulating factor. Naringin, at a concentration of 1, 10, 50, and 100 μg/mL, was respectively added to the medium. Seven days later, the osteoclasts were determined through tartrate-resistant acid phosphatase (TRAP) staining. Mature osteoclasts were isolated from newborn rabbits and cultured for three days on bone slices. Naringin at a concentration of 1, 10, 50, and 100 μg/mL was respectively added to the medium. The resorption bone slices were quantified, and the area was calculated after toluidine blue and Mayer-hematoxylin staining. Polymethyl methacrylate (PMMA) particles were implanted on the calvariae of C57BL/J6 mice. Naringin, at a dose of 50 μg/kg and 100 μg/kg, was respectively given intraperitoneally for seven. Seven days later, the calvariae were removed and processed for pathological analysis. The result indicated that naringin treatment effectively inhibited in vitro osteoclastogenesis and inhibited mature osteoclasts. In vivo data indicated that naringin strongly inhibited PMMA-induced osteolysis. Naringin can effectively inhibit osteoclastogenesis and suppress wear particles-induced osteolysis and might be useful in the treatment or prevention of wear particles-induced osteolysis and aseptic loosening for its effect on osteoclast generation and function.

Polarized osteoclasts put marks of tartrate-resistant acid phosphatase on dentin slices — A simple method for identifying polarized osteoclasts

Osteoclasts form ruffled borders and sealing zones toward bone surfaces to resorb bone. Sealing zones are defined as ringed structures of F-actin dots (actin rings). Polarized osteoclasts secrete protons to bone surfaces via vacuolar proton ATPase through ruffled borders. Catabolic enzymes such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K are also secreted to bone surfaces. Here we show a simple method of identifying functional vestiges of polarized osteoclasts. Osteoclasts obtained from cocultures of mouse osteoblasts and bone marrow cells were cultured for 48h on dentin slices. Cultures were then fixed and stained for TRAP to identify osteoclasts on the slices. Cells were removed from the slices with cotton swabs, and the slices subjected to TRAP and Mayer's hematoxylin staining. Small TRAP-positive spots (TRAP-marks) were detected in the resorption pits stained with Mayer's hematoxylin. Pitted areas were not always located in the places of osteoclasts, but osteoclasts existed on all TRAP-marks. A time course experiment showed that the number of TRAP-marks was maintained, while the number of resorption pits increased with the culture period. The position of actin rings formed in osteoclasts corresponded to that of TRAP-marks on dentin slices. Immunostaining of dentin slices showed that both cathepsin K and vacuolar proton ATPase were colocalized with the TRAP-marks. Treatment of osteoclast cultures with alendronate, a bisphosphonate, suppressed the formation of TRAP-marks and resorption pits without affecting the cell viability. Calcitonin induced the disappearance of both actin rings and TRAP-marks in osteoclast cultures. These results suggest that TRAP-marks are vestiges of proteins secreted by polarized osteoclasts.

Identification of apoptotic proteins in thyroid gland from patients with Graves' disease and Hashimoto's thyroiditis

Apoptosis, i.e. natural programmed cell death, is a physiological phenomenon indispensable for normal functioning of the organism. The signal to apoptosis can be started practically in any cell. Disturbances in the apoptosis regulation determine the essential link of the pathogenesis of many diseases, including autoimmune thyroid disorders.The aim of the study was to assess the expression of Fas/FasL and caspase eight in the tissues of the thyroid gland in patients with Graves' disease (GD), non-toxic nodular goiter (NTNG) and Hashimoto's thyroiditis (HT). The analysis of Fas/FasL expression was performed by western blot and immunohistochemical investigation with DAB-visualization and Mayer's hematoxylin staining. Caspase-8 expression in thyroid follicular cells was assayed by western blot method.Identification of the proapoptotic proteins FasL and Fas exhibited their pronounced expression in the thyroid tissue in GD patients (++; ++) and HT (+++; +++) as compared to the NTNG group (0/+; 0/+). Among the study groups, the expression of caspase-8 was revealed in band 55 kDa from patients with autoimmune thyroid diseases.In GD patients, the percentage of thyrocytes with FasL expression correlated positively with TRAb (R = 0.58, p < 0.02). However, no such correlations were noted in HT or non-toxic multinodular goiter. There were no significant correlations between thyroid hormones and the percentage of thyrocytes with Fas and FasL expression.In conclusion, our findings suggest that the changes in the expression of apoptotic molecules on the surface of T lymphocytes and thyroid follicular cells in patients with autoimmune thyroid disorders reflect their substantial involvement in the pathogenesis of GD and HT. In addition, analysis of Fas/FasL and caspase-8 expression in thyroid tissue may indicate the disease activity and immunological phenotype.

Inhibiting wear particles-induced osteolysis with doxycycline

To study the effect of doxycycline (DOX) on osteoclastogenesis, mature osteoclast fate and function, wear particles-induced osteoeolysis, and to provide some foundation for treating aseptic loosening and osteolysis after joint arthroplasty. Osteoclasts were generated from mouse bone marrow monocytes with the receptor activator of NF-kappaB ligand and the macrophage colony stimulating factor. DOX at a concentration of 5, 10, 15, and 20 microg/mL was respectively added to the medium. Seven days later, the osteoclasts were determined through tartrate-resistant acid phosphatase (TRAP) staining. Mature osteoclasts were isolated from newborn rabbits and cultured for 3 d in 24-well plates or on bone slices. DOX at a concentration of 5, 10, 15, and 20 microg/mL was respectively added to the medium. After TRAP staining, the osteoclasts were counted, resorption on bone slices was quantified, and the area was calculated after toluidine blue and Mayer-hematoxylin staining. Polymethyl methacrylate (PMMA) or ultra-high molecular weight polyethylene (UHMWPE) particles were implanted on the calvariae of C57BL/J6 mice. DOX, at a dose of 2 and 10 mg x kg(-1) x d(-1), was respectively given intraperitoneally for 7 d. Seven days later, the calvariae were removed and processed for pathological analysis. DOX treatment effectively inhibited in vitro osteoclastogenesis, affected the fate of mature osteoclasts, and inhibited mature osteoclasts, causing bone resorption. In vivo data indicated that DOX strongly inhibited PMMA or UHMWPE-induced osteolysis and osteoclastogenesis. DOX can effectively inhibit osteoclastogenesis and affect mature osteoclast fate and suppress wear particles induced by osteolysis and osteoclastogenesis. DOX might be useful in the treatment or prevention of wear particles-induced osteolysis and aseptic loosening for its effect on osteoclast generation and mature osteoclast fate and function.

Preliminary study of vascular mimicry in pterygium

To investigate the role of vascular mimicry in the development of the pterygium. Two hundred and eighteen pterygium specimens were collected from pterygium operations, 97 of them were studied under the light microscope by immunohistochemistry and immunohistochemistry combined with PAS stain (primary antibodies included CD34, laminin and VEGF). Specimens were incubated with primary antibodies at 37 degrees C for 60 mins, followed by incubation with secondary antibodies at 37 degrees C for 30 mins, stained by DAB, oxidized by 0.5% periodic acid for 10 mins, stained by Schiff staining for 20 mins, counterstained by Mayer-Hematoxylin staining for 3 mins, washed with water and then mounted. Vascular mimicry was identified in 97 of 218 pterygium specimens, which appeared as tubular type and network matrix type. The septa of meshwork stained positively with PAS stain. CD34 immunohistochemical staining combined with PAS staining showed that the arcs and cells in the meshwork were stained positively with CD34 and were encircled by PAS positive extracellular matrix. Laminin immunohistochemical staining combined with PAS staining showed that part of endothelial cells and blood vessel walls were stained positively by laminin, but most cells in the tubular-like region were stained negatively. VEGF immunohistochemical staining combined with PAS staining showed that fibroblast-like cells in the network matrix were stained positively by VEGF and the septa of meshwork and extracellular matrices were stained positively by PAS. Vascular mimicry exists in the pterygium and is one of the blood supply pathways of the pterygium.

Modification of the Mayer Hematoxylin Stain

AbstractA modified Mayer hematoxylin stain that can be combined with either phloxinelsaffron or eosin for general staining 01 as a counterstain is presented. This method uses 10% less alum, and chloral hydrate is replaced by absolute ethanol, a more effective penetrator and bacteriostat. Phenol is recommended for formalin pigment removal in formaldehyde fixed tissues to effect a more esthetically pleasing section with most techniques. Saffron bought through the health food stores is 8 times less expensive than that bought commercially. (The J Histotechnol 14:163, 1991)

Modification of the Mayer Hematoxylin Stain

Modification of the Mayer Hematoxylin Stain