BACKGROUND: Though elastic fibers are as important as collagen fibers in interpretation of the histopathologic findings, it is impossible to observe them on the hematoxylin & eosin (H&E) stained specimen. OBJECTIVE: Characterizing eosin fluorescence emitted by elastic fibers in H&E stained specimens. METHODS: Normal skin tissue sections were stained in 4 different ways (unstained, hematoxylin only, eosin only, H&E) and observed under a fluorescence microscope using a FITC filter set. Fluorescent findings of 30 H&E-stained specimens showing abnormal dermal findings were compared with bright field findings of Miller's elastic stained specimen. RESULTS: Strong eosin fluorescence was related to the differential binding property of eosin with elastic fibers. Hematoxylin stain quenched excessive eosin fluorescence from other tissue components and contributed to better contrast. Fluorescence microscopy of H&E-stained sections was found to be especially useful in observing mature elastic fibers in the reticular dermis. In 74% of the specimens, eosin fluorescence findings of elastic fibers in reticular dermis matched well with that of specimens with elastic fiber special stain. CONCLUSION: Analysis of skin elastic fibers by fluorescence microscopy is a useful and complementary method to reveal hidden elastic fibers in H&E-stained specimens.
Collagen ; Dermis ; Elastic Tissue ; Enzyme Multiplied Immunoassay Technique ; Eosine Yellowish-(YS) ; Fluorescein-5-isothiocyanate ; Fluorescence ; Hematoxylin ; Microscopy, Fluorescence ; Skin

It is known that there are numerous chemotatic secretoneurin -immunoreactive nerve fibers and movable MHC class II -immunoreactive dendritic cells in the normal uterine cervix. And the relationships between them are not fully understood. The aim of this study is to reveal that secretoneurin could give to chemotatic influence to dendritic cells in inflammational state. Virgin female Sprague -Dawley rats (n = 20; approximately 2 months old; 200 ~250 g body weight) were used in this study. Animals (n = 10) were injected with 5% formalin (0.5 ml/day, 5 days) in experiment group. Animals were deeply anesthetized with 3.5% chloral hydrate (100 mg/kg, i.p.) and uterine cervix were removed. Immunostaining was done according to standard methods used routinely. In brief, tissue sections were incubated with primary antibodies generated in mouse anti -rat MHC class II antibody and mouse or rabbit anti -rat secretoneurin antibody for single and double immunostains. FITC for secretoneurin and rhodamine for MHC class II were used as secondary antibodies in double stains. Tissue sections were observed by using light and confocal laser scanning microscopes. The results were as follows; 1. Numerous secretoneurin -immunoreactive nerve fibers were located in the lamina propria and those were not found in the epithelium of normal rat uterine cervix. 2. MHC class II -immunoreactive dendritic cells were mainly located in the epithelium and the lamina propria of normal rat uterine cervix. 3. On the inflammation state, MHC class II -immunoreactive dendritic cells were mainly located in the lamina propria and those were not found in the epithelium of rat uterine cervix. According to above results, it is suggested that secretoneurin can give to chemotatic influence to dendritic cells in inflammational state. Therefore, secretoneurin is considered to be used for dendritic cell immunotheraphy.
Animals ; Antibodies ; Cervix Uteri ; Chloral Hydrate ; Coloring Agents ; Dendritic Cells ; Epithelium ; Female ; Fluorescein-5-isothiocyanate ; Formaldehyde* ; Humans ; Infant ; Inflammation ; Mice ; Mucous Membrane ; Nerve Fibers ; Rats* ; Rhodamines ; Uterus*

OBJECTIVETo investigate the dose and time-dependent effects of lipopolysaccharide (LPS) on cytoskeletal F-acitn and G-actin reorganizations by visualizing their distribution and measuring their contents in human umbilical vein endothelial cell line ECV-304.

METHODSF-actin was labeled with rhodamine-phalloidin and G-actin with deoxyribonuclease I (DNase I)conjugated with fluorescein isothiocyanate (FITC). Contents of cytoskeletal proteins were obtained by flow cytometry.

RESULTSF-actin was mainly distributed peripherally in endothelial cells under normal conditions. LPS stimulation caused the formation of stress fibers and filopodia. G-actin was normally seen in perinuclear and nuclear areas in control ECV-304 cells. Under LPS stimulation, G-actin dots appeared in the cytoplasmic region. The actin disorganization was accompanied by the time- and dose- dependent decrease in F-actin pool and increase in G-actin pool.

CONCLUSIONSLPS can induce characteristic morphological alterations of actin cytoskeleton and formation of intercellular gap in endothelial cells, accompanied by changes in F-actin and G-actin pools.

Actins ; drug effects ; Analysis of Variance ; Cells, Cultured ; Deoxyribonuclease I ; Dose-Response Relationship, Drug ; Endothelial Cells ; chemistry ; Escherichia coli ; Fluorescein-5-isothiocyanate ; Fluorescent Dyes ; Humans ; Lipopolysaccharides ; pharmacology ; Phalloidine ; Rhodamines ; Umbilical Veins ; cytology

The present study aimed to determine the role of Rho/Rho kinase (Rho/ROCK) phosphorylation on advanced glycation end products (AGEs)-induced morphological and functional changes in human dermal microvascular endothelial cells (HMVECs). HMVECs were respectively incubated with different concentrations of AGEs-modified human serum albumin (AGEs-HSA) for different time. In some other cases, HMVECs were pretreated with ROCK inhibitors (H-1152 or Y-27632). The morphological changes of F-actin cytoskeleton were visualized by rhodamine-phalloidin staining and the phosphorylation of Rho and ROCK were determined by Western blot. Endothelial monolayer permeability was assessed by measuring the flux of FITC-albumin across the endothelial cells. The results showed that the distribution of F-actin was significantly altered by AGEs-HSA in time and dose-dependent patterns. These effects were inhibited by ROCK inhibitors. The phosphorylation of Rho and RCOK was remarkably increased by AGEs-HSA treatment while total Rho and ROCK protein levels were not affected. The permeability of endothelial monolayer was dramatically increased by AGEs-HSA, and both ROCK inhibitors (H-1152 or Y-27632) attenuated these hyperpermeability responses. The results obtained suggest that the phosphorylation of Rho/ROCK plays an important role in AGEs-induced morphological and functional alterations in HMVECs.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; pharmacology ; Actin Cytoskeleton ; metabolism ; Actins ; metabolism ; Amides ; pharmacology ; Endothelial Cells ; metabolism ; Endothelium, Vascular ; cytology ; Fluorescein-5-isothiocyanate ; analogs & derivatives ; metabolism ; Glycation End Products, Advanced ; pharmacology ; Humans ; Phalloidine ; analogs & derivatives ; Phosphorylation ; Pyridines ; pharmacology ; Rhodamines ; Serum Albumin ; metabolism ; pharmacology ; Serum Albumin, Human ; Signal Transduction ; rho-Associated Kinases ; metabolism

Natural cytotoxicity mediated by natural killer (NK) cells is believed to play an important role in host anticancer defense mechanisms. The aim of this study is to compare the number of NK cells in patients with colorectal cancer and hemorrhoids, and before and after surgery in patients with colorectal cancer. Twenty colorectal cancer patients and twenty hemorrhoid ones were studied. Venous blood samples were obtained preoperatively, and on the 7th, and 14th postoperative days. Mononuclear cells were isolated over Ficoll-Hypaque gradients, and T cells, B cells, and NK cells were measured with CD3 FITC (T cell), CD 19 PE (B cell), and CD56 FITC (NK cell) antibody, The number of T cell (/mm3) was 1224, 1280, and 1125 at preoperative, 7th, and 14th postoperative day in hemorrhoid patients and 1195, 901, and 1060 in colorectal cancer patients respectively. The number of B cell (/mm3) was 243, 160, and 250 in hemorrhoid patients and 147, 78, and 113 in colorectal cancer patients. The number NK cell (/mm3) was 148, 156, and 143 in hemorrhoid patients and 129, 85, and 128 in colorectal cancer patients. There was no difference among Dukes stages in the number of NK cells. In conclusion, the number of NK cells was not changed in colorectal cancer patients compared with hemorrhoid ones. Major operation changed the number of NK cells in colorectal cancer patients.
B-Lymphocytes ; Colorectal Neoplasms* ; Defense Mechanisms ; Fluorescein-5-isothiocyanate ; Hemorrhoids ; Humans ; Killer Cells, Natural* ; T-Lymphocytes

Author investigated the deposition of immunoglobulins and complements in thc skin of 56 patients with 19 various dermatoses by direct immunofluorescent (DIF) staining. The biopsied specimens were quick-frozen(by dry ice-acetone) and stained with FITC conjugated antihuman immunoglobulin and complement after cutting in a cryostat at -20C~30C. (countinued...)
Complement System Proteins ; Dronabinol ; Fluorescein-5-isothiocyanate ; Humans ; Immunoglobulins ; Skin ; Skin Diseases*

AIMTo establish a simple and effective method for RBCs labeling and survival assays, and the qualities of rabbit RBCs preserved in GMA solution at 4 degrees C were verified.

METHODSThe bloods were taken through the ear arteries of the rabbits. The RBCs were labeled by fluorescein isothiocyanate (FITC), and were reinjected to the same rabbit through ear veins. The percentage of FITC labeled RBCs was assayed by FACS at a series of times after injection. The SAS software was employed to analyze the data and establish the regression equations. The 24-hour recovery and the half-life span of the labeled RBCs were calculated according to the equations.

RESULTSThe 24-hour recovery and the half-life span of the labeled RBCs in the control group were 93.76% +/- 5.40% and 22.50% +/- 4.37 days respectively, which was in agreement with the previous papers. The 24-hour recovery and the half-life span of the labeled RBCs in the GMA group were 89.13% +/- 7.10% and 11.41% +/- 1.63 days respectively, which was coincident with the infusion conditions.

CONCLUSIONCompared with other methods of RBCs labeling in vivo, FITC labeling was thought to be easier and cheaper to use, which could facilitate the analysis of the biological character of the labeled cells, and could be used to trace the fate of labeled cells.

Animals ; Blood Preservation ; methods ; Erythrocyte Aging ; physiology ; Erythrocyte Count ; Erythrocytes ; physiology ; Fluorescein-5-isothiocyanate ; Rabbits ; Software

The objective of this work is to evaluate the effect of polyamidoamine (PAMAM) dendrimers on electroosmotic flow (EOF) through skin. The effect of size and concentration of dendrimer was studied, using generation 1, 4 and 7 dendrimer (G1, G4 and G7, respectively). As a marker molecule for the direction and magnitude of EOF, a neutral molecule, acetoaminophen (AAP) was used. The visualization of dendrimer permeation into the current conducting pore (CCP) of skin was made using G4–fluorescein isothiocyanate (FITC) conjugate and confocal microscopy. Without dendrimer, anodal flux of AAP was much higher than cathodal or passive flux. When G1 dendrimer was added, anodal flux decreased, presumably due to the decrease in EOF by the association of G1 dendrimer with net negative charge in CCP. As the generation increased, larger decrease in anodal flux was observed, and the direction of EOF was reversed. Small amount of methanol used for the preparation of dendrimer solution also contributed to the decrease in anodal flux of AAP. Cross-sectional view perpendicular to the skin surface by confocal laser scanning microscope (CLSM) study showed that G4 dendrimer-FITC conjugate (G4-FITC) can penetrate into the viable epidermis and dermis under anodal current. The permeation route seemed to be localized on hair follicle region. These results suggest that PAMAM dendrimers can permeate into CCP and change the magnitude and direction of EOF. Overall, we obtained a better understanding on the mechanistic insights into the electroosmosis phenomena and its role on flux during iontophoresis.
Acetaminophen ; Dendrimers* ; Dermis ; Electroosmosis* ; Epidermis ; Fluorescein-5-isothiocyanate ; Hair Follicle ; Iontophoresis ; Methanol ; Microscopy, Confocal ; Skin*

In order to prepare angiopep-2 modified fluorescein isothiocyanate-labeled neurotoxin nanoparticles( ANG-NPs/FITCNT),emulsion/solvent evaporation method was used with m PEG-PLA and ANG-PEG-PLA( in proper proportions) as carriers and with FITC-NT as drug. With particle size and encapsulation efficiency as comprehensive indexes,the effects of different ultrasound power and ultrasound time combinations on the process were investigated. The in vitro release characteristics of nanoparticles in PBS buffer at p H 7. 4 and p H 6. 5 were investigated by dialysis method. The results indicated that the optimum process for preparing ANG-NPs/FITC-NT was as follows: ultrasonic power 90 W,ultrasonic time 30 s. In such optimal process,ANG-NPs/FITC-NT were well-shaped under the transmission electron microscope,with an average particle size of( 123. 9±0. 5) nm,Zeta potential of(-10. 5±0. 5) m V,encapsulation efficiency of( 68. 1±0. 4) %,and the drug loading of( 0. 82±0. 01) %. The in vitro drug release profiles of the nanoparticles in PBS buffer at p H 7. 4 and p H 6. 5 were both consistent with Ritger-Peppas equation,ln Q = 0. 508 8 lnt-2. 285 0,r = 0. 961 5( p H 7. 4) and ln Q= 0. 449 9 lnt-1. 855 3,r = 0. 970 3( p H 6. 5),respectively. The experiment results proved that the nanoparticles prepared by emulsion/solvent evaporation method had uniform particle size,high encapsulation efficiency and in vitro sustained release characteristic,which might be a potential carrier for NT intracerebral drug delivery.
Drug Carriers ; Fluorescein-5-isothiocyanate ; Nanoparticles ; Particle Size ; Peptides ; Polyethylene Glycols

PURPOSE: This study was performed to evaluate the parameters of tear function and ocular surface in patients with pinguecula and pterygium. METHODS: The corneal sensitivity test (CST), tear break-up time (BUT), basal tear secretion test, fluorescein staining, rose bengal staining, tear clearance test, and conjunctival impression cytology were evaluated in patients with unilateral pinguecula and pterygium. The results were also evaluated according to the severity of pterygium. RESULTS In patients with pinguecula, BUT (P=0.03) and goblet cell density (P<0.01) were decreased, while the rose bengal staining score (P=0.01) was increased significantly. In patients with pterygium, CST (P=0.01), BUT (P<0.01), and goblet cell density (P<0.01) decreased, and the fluorescein staining score (P<0.01), rose bengal staining score (P<0.01) and grade of conjunctival metaplasia (P<0.01) increased significantly. In comparison with mild pterygium, moderate pterygium demonstrated decreased CST (P=0.01) and BUT (P=0.01), and an increased rose bengal staining score (P=0.04). CONCLUSIONS: The tear films and ocular surface changes in patients with pinguecula and pterygium are worse than in control group. Also, there is close correlation between the severity of pterygium and dry eye condition.
Fluorescein ; Goblet Cells ; Humans ; Metaplasia ; Pinguecula* ; Pterygium* ; Rose Bengal ; Tears*