Genetic code expansion (GCE) allows the incorporation of unnatural amino acids into proteins via using stop codons. GCE may achieve site-specific labeling of proteins in combination with the click reaction. Compared with other labeling tools such as fluorescent proteins and tagged antibodies, the compound molecules used in protein labeling by GCE technology are smaller, and therefore, may less interfere the conformational structure of proteins. In addition, through click reaction, GCE allows a 1:1 stoichiometric ratio of the target protein molecule and the fluorescent dye, and the protein can be quantified based on the fluorescence intensity. Thus, GCE technology has great advantages in the researches that require the exposition of living cells under high laser power for longer time, for example, in the context of single molecule tracing and super-resolution microscopic imaging. Meanwhile, this technology lays the foundation for improving the accuracy of positioning and molecule counting in the imaging process of living cells. This review summarized the GCE technology and its recent applications in functionally characterizing, labeling and imaging of proteins.
Amino Acids/chemistry* ; Fluorescent Dyes/chemistry* ; Genetic Code ; Proteins/chemistry*

OBJECTIVETo explore a method for combining Fluoro-Jade B (FJB) staining with immunofluorescent staining in rats with focal cortical infarction.

METHODPermanent distal middle cerebral artery occlusion (dMCAO) was induced in rats by electrocoagulation. The rat models were randomized into two groups, and frozen sections of the brain tissues from each group were stained with FJB followed by immunofluorescent staining or in the reverse order.

RESULTSFJB staining followed by immunofluorescence staining clearly visualized both FJB-positive and immunofluorescence-positive cells in the frozen sections, but the staining protocol in the reverse sequence failed to clearly show the immunofluorescence-positive cells.

CONCLUSIONFJB staining prior to immunofluorescence staining does not affect the staining effect of protein immunofluorescent staining and better visualizes the positive cells.

Animals ; Brain ; pathology ; Fluoresceins ; chemistry ; Fluorescent Antibody Technique ; methods ; Fluorescent Dyes ; chemistry ; Infarction, Middle Cerebral Artery ; Rats ; Staining and Labeling ; methods

Fumonisin B1 (FB1) is a carcinogenic mycotoxin found in commodities such as corn and corn-originated products. An aptamer-based method for detection of FB1 was developed using the fluorescent dye PicoGreen, which can recognize and bind double-stranded DNA. A peak fluorescence of PicoGreen was obtained in 15 min in the presence of FB1 aptamer, which formed a double-stranded hybridizer DNA with its complementary strand. The excitation and emission wavelengths for PicoGreen detection were 480 nm and 520 nm, respectively. The sensitivity of this aptamer/PicoGreen-based method was 0.1 μg/L. This method showed a good linearity for FB1 concentration ranging from 0.1 to 1 μg/L. The entire detection procedure for FB1 could be completed within 40 min. No cross reactions were observed with any other mycotoxins against aflatoxin B1, ochratoxin A, citrinin and zearalenone, demonstrating high specificity towards FB1 aptamer. Agreement between commercial, antibody-based enzyme-linked immunosorbent assay (ELISA) kit and aptamer method was excellent with a kappa value of 0.857. Taken together, this aptamer/PicoGreen-based method is more cost-effective, time-saving and useful than ELISA for detection of FB1.
Aflatoxin B1 ; Enzyme-Linked Immunosorbent Assay ; Fluorescence ; Fluorescent Dyes ; chemistry ; Fumonisins ; analysis ; Mycotoxins ; analysis ; Ochratoxins ; Organic Chemicals ; chemistry ; Staining and Labeling ; Zea mays

We prepared protoplasts from Salvia miltiorrhiza Bunge suspension culture cells. Then, the protoplasts' vitality and functions were tested by fluorescein diacetate staining method and Fluo-3/AM flourescent probe. The optimal condition of protoplast isolation was Cellulase R-10 1.5%, Pectinase Y-23 0.3%, Macerozyme R-10 0.5%, 40 r/min 12 h, 600 r/min 5 min, and the protoplasts yield was 1.1x10(6) cells/g FW, the vitality was more than 95% by using fluorescein diacetate staining method. It has been confirmed that calcium fluorescent probe Fluo-3/AM can be successfully loaded into protoplasts.
Aniline Compounds ; chemistry ; Cell Culture Techniques ; Cellulase ; chemistry ; Fluorescent Dyes ; chemistry ; Protoplasts ; chemistry ; Salvia miltiorrhiza ; growth & development ; Xanthenes ; chemistry

As a new type of carbon nanomaterials, fluorescent carbon dots (fluorescent CDs) have many advantages when compared with the traditional fluorescent probes. They are photoluminescence stable and resistance to photo bleaching. Moreover, they are excellent in biocompatibility, low-toxic and easy to modify. All these above make them a promising optical image material as a probe in optical image. This article reviews structure, the common carbon sources, the preparation methods, and the light-emitting principles of the carbon dots. We also introduce the research progress of fluorescent carbon dots in biomedicine, and the problems need to be resolved in the study of fluorescent CDs.
Carbon ; chemistry ; Fluorescent Dyes ; chemistry ; Nanostructures ; chemistry ; Quantum Dots ; chemistry

In this study, an approach based on triple-color fluorescence probes was developed for screening potential nephro-protective bioactive substances. Three fluorescent probes (i. e. FDA, MTR and Hoechst 33342) were used to label HK-2 cells injured by doxorubicin hydrochloride, and cellular fluorescence images were subsequently acquired and analyzed by a cellular-fluorescence image microscopy platform. The established method was applied to screening 53 components of Carthami Flos, and three components C17, C18 and C19 were found to exhibit nephroprotective effects against doxorubicin hydrochloride induced injury on HK-2 cells. Eight compounds (i. e. hydroxysafflor yellow A, 6-hydroxykaempferol-3-O-rutinoside-6-O-glucoside, 6-hydroxykaempferol-3,6-di-O-gluco-side or 6-hydroxykaempferol-6, 7-di-O-glucoside, 6-hydroxykaempferol-3-O-rutinoside, 6-hydroxykaempferol-3-O-glucoside or 6-hydroxykaempferol-7-O-glucoside, rutin, isoquercetin, and kaempferol-3-O-rutinoside) in components C17, C18 and C19 were preliminarily identified by liquid chromatography-mass spectrometry (LC-MS). Isoquercetin, rutin, kaempferol-3-O-rutinoside, and hydroxysafflor yellow A were confirmed by comparing with reference substances, Further study indicated that these four compounds had moderate nephroprotective effects, while isoquercetin showed a significant nephroprotective effect in a dose-dependent manner. These results suggest that isoquercetin, rutin, kaempferol-3-O-rutinoside and hydroxysafflor yellow A might be the nephroprotective bioactive substances in Carthami Flos.
Carthamus ; chemistry ; Cell Line ; Chromatography, High Pressure Liquid ; Dose-Response Relationship, Drug ; Drug Evaluation, Preclinical ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Flowers ; chemistry ; Fluorescent Dyes ; chemistry ; Humans ; Kidney ; chemistry ; cytology ; drug effects ; Protective Agents ; chemistry ; pharmacology

In this paper, Liuwei Dihuang pill was used to study the identification of Chinese patent medicine by fluorescence sequencing typing technology. The DNA of Paeonia suffruticosa was used as template to amplify by five pair of FAM fluorescence labeling primers. Then, the amplified products were sequenced. The sequencing results were analyzed by GeneMarker V1.80 to screen the best fluorescence labeling primers. As a result, psbA-trnH fluorescence labeling primer was used to identify the raw materials of Liuwei Dihuang pill. The results showed that three kinds of raw plant medicinal materials in Liuwei Dihuang pill were able to be correctly identified by psbA-trnH fluorescence labeling primer. The fluorescence sequencing typing technology can stably and accurately distinguish raw medicinal materials in Chinese patent medicine.
DNA Primers ; chemistry ; genetics ; DNA, Plant ; chemistry ; genetics ; Drugs, Chinese Herbal ; chemistry ; standards ; Fluorescent Dyes ; chemistry ; Plants, Medicinal ; chemistry ; genetics ; Polymerase Chain Reaction ; instrumentation ; methods ; Quality Control ; Staining and Labeling

Astragalus polysaccharides was lounded to 4-(2-aminoethylphenol), followed by labeling the APS-Tyr with fluorescein-5-isothiocyanate (FITC) at the secondary amino group. The absorption enhancement effects of low molecular weight chitosan and protamine on astragalus polysaccharides were evaluated via Caco-2 cell culture model. The results show that the fluorecent labeling compound has good stability and high sensitivity. On the other hand low molecular weight chitosan and protamine also can promoted absorption of the astragalus polysaccharides without any cytotoxity, and the absorption increase was more significant with increasing the amount of low molecular weight chitosan and protamine. At the same time, the low molecular weight chitosan has slightly better effect. The transepithelial electric resistance (TEER) of Caco-2 cells show that absorption enhancers could improve its membrane transport permeability by opening tight junctions between cells and increasing the cell membrane fluidity.
Absorption ; Astragalus Plant ; chemistry ; Biological Transport ; Caco-2 Cells ; Fluorescein-5-isothiocyanate ; chemistry ; Fluorescent Dyes ; chemistry ; Humans ; Plant Extracts ; chemistry ; pharmacokinetics ; Polysaccharides ; chemistry ; pharmacokinetics

Relatively uniform-sized nanoparticles made of poly (lactic-co-glycolic acid) (PLGA) were prepared by premix membrane emulsification method. After the drug loading property was completed, the dynamic tissue distribution of nanoparticles was recorded. With the average particle size and span as indexes, membrane pore size, number of passing membrane times, membrane pressure, volume ratio of oil-water phase and the concentration of poly(vinyl alcohol) (PVA) in external water phase were investigated by single factor test, the optimum preparation technology of blank PLGA nanlparticles was as following: pore size of SPG membrane was 1 μm, membrane pressure was 1. 15 MPa, the number of passing membrane time was 3, the mass fraction of PVA of 2%, volume ratio of oil-water phase of 1 : 5. Prepared nanoparticles were round with smooth surface, the mean diameter was 332.6 nm, span was 0.010, the confocal laser scanning microscope (CLSM) concluded that fluorescent substance is uniform composizion in PLGA nanoparticle, and the in vivo imaging technology in mice include that the nanoparticles show good liver and spleen targeting property.
Animals ; Drug Carriers ; chemistry ; Drug Delivery Systems ; instrumentation ; Emulsions ; chemistry ; Fluorescent Dyes ; chemistry ; Lactic Acid ; chemistry ; Mice ; Mice, Nude ; Nanoparticles ; chemistry ; Particle Size ; Polyglycolic Acid ; chemistry

Animals ; Cattle ; Cell Line ; Electrophoresis, Polyacrylamide Gel ; Fluorescent Dyes ; chemistry ; Malondialdehyde ; chemistry ; Mice ; Microscopy, Confocal ; Serum Albumin, Bovine ; analysis ; chemistry ; metabolism ; Ultraviolet Rays