Staining-free gel electrophoresis-based multiplex enzyme assay using DNA and peptide dual-functionalized gold nanoparticles

We report a simple staining-free gel electrophoresis method to simultaneously probe protease and nuclease. Utilizing gold nanoparticles (Au-NPs) dual-functionalized with DNA and peptide, the presence and concentration of nuclease and protease are determined concurrently from the relative position and intensity of the bands in the staining-free gel electrophoresis. The use of Au-NPs eliminates the need for staining processes and enables naked eye detection, while a mononucleotide-mediated approach facilitates the synthesis of DNA/peptide conjugated Au-NPs and simplifies the operation procedures. Multiplex detection and quantification of DNase I and trypsin are successfully demonstrated.     

显微红外光谱法研究染色处理的口腔组织切片

本文用显微红外光谱法研究了经染色处理和未经染色处理口腔正常组织和肿瘤组织的显微红外光谱。实验结果表明，对样品进行染色处理并未对上述口腔组织的特征光谱产生明显的影响。这就为深化认识人体正常组织和肿瘤组织的结构与光谱关系创造了机会。     

A valve‐based microfluidic device for on‐chip single cell treatments

Assays toward single‐cell analysis have attracted the attention in biological and biomedical researches to reveal cellular mechanisms as well as heterogeneity. Yet nowadays microfluidic devices for single‐cell analysis have several drawbacks: some would cause cell damage due to the hydraulic forces directly acting on cells, while others could not implement biological assays since they could not immobilize cells while manipulating the reagents at the same time. In this work, we presented a two‐layer pneumatic valve‐based platform to implement cell immobilization and treatment on‐chip simultaneously, and cells after treatment could be collected non‐destructively for further analysis. Target cells could be encapsulated in sodium alginate droplets which solidified into hydrogel when reacted with Ca²⁺. The size of hydrogel beads could be precisely controlled by modulating flow rates of continuous/disperse phases. While regulating fluid resistance between the main channel and passages by the integrated pneumatic valves, on‐chip capture and release of hydrogel beads was implemented. As a proof of concept for on‐chip single‐cell treatments, we showed cellular live/dead staining based on our devices. This method would have potential in single cell manipulation for biochemical cellular assays.     

Staining methods applied to glycol methacrylate embedded tissue sections

The use of glycol methacrylate (GMA) avoids some technical artifacts, which are usually observed in paraffin-embedded sections, providing good morphological resolution. On the other hand, weak staining have been mentioned during the use of different methods in plastic sections. In the present study, changes in the histological staining procedures have been assayed during the use of staining and histochemical methods in different GMA-embedded tissues.

Samples of tongue, submandibular and sublingual glands, cartilage, portions of respiratory tract and nervous ganglion were fixed in 4% formaldehyde and embedded in glycol methacrylate. The sections of tongue and nervous ganglion were stained by H&E. Picrosirius, Toluidine Blue and Sudan Black B methods were applied, respectively, for identification of collagen fibers in submandibular gland, sulfated glycosaminoglycans in cartilage (metachromasia) and myelin lipids in nervous ganglion. Periodic Acid-Schiff (PAS) method was used for detection of glycoconjugates in submandibular gland and cartilage while AB/PAS combined methods were applied for detection of mucins in the respiratory tract. In addition, a combination of Alcian Blue (AB) and Picrosirius methods was also assayed in the sublingual gland sections.

The GMA-embedded tissue sections showed an optimal morphological integrity and were favorable to the staining methods employed in the present study. In the sections of tongue and nervous ganglion, a good contrast of basophilic and acidophilic structures was obtained by H&E. An intense eosinophilia was observed either in the striated muscle fibers or in the myelin sheaths in which the lipids were preserved and revealed by Sudan Black B. In the cartilage matrix, a strong metachromasia was revealed by Toluidine Blue in the negatively-charged glycosaminoglycans. In the chondrocytes, glycogen granules were intensely positive to PAS method. Extracellular glycoproteins were also PAS positive in the basal membrane and in the region occupied by the lamina externa and reticular fibers surrounding each smooth muscle cells of the blood vessels. In the epithelial cells of the respiratory tract, acid and neutral mucins were histochemically detected by AB and PAS methods, respectively. Moreover, granules containing acid and neutral mucins were revealed in purple by AB and PAS concomitantly. In the sublingual gland sections, a distinct affinity of acid mucins by AB (in turquoise-blue) and collagen fibers by Picrosirius (in red) was obtained when these methods were combined. Although some routine dyes used in paraffin sections have showed a weak stain in historesin sections, our results showed that different dyes could be applied in GMA sections if modified staining procedures were assayed. Therefore, appropriate staining contrast and, thus, detection of one or different substances in a same section can be acquired in association to the good morphological resolution provided by GMA.     

Polyacrylamide gel electrophoresis separation and detection of polyamidoamine dendrimers possessing various cores and terminal groups

Detection and separation of polyamidoamine dendrimers possessing various cores and surface groups was studied by polyacrylamide gel electrophoresis. Although many dyes and staining techniques were able to detect dendrimers on polyacrylamide gels, Coomassie Blue was found to be the most sensitive and convenient. Amine and hydroxyl terminated dendrimers were best separated under acidic conditions, while dendrimers with carboxyl surfaces required alkaline buffers. Some dendrimers were very susceptible to diffusion that could occur during their separation, staining or destaining steps. In the absence of an appropriate fixation step, dendrimers could be resolved by using small pore size gels and low voltage or current. Increasing core lengths did not significantly affect migration of a given dendrimer generation but exhibited improved separation and staining characteristics. Polyacrylamide gel electrophoresis was found to be a rapid, inexpensive, and reliable procedure to characterize many different water-soluble dendritic macromolecules.     

A simple,time‐saving,microwave‐assisted periodic acid–Schiff´s staining of glycoproteins on 1D electrophoretic gels

We introduce an optimized periodic acid–Schiff´s staining of glycoproteins on 1D electrophoretic gels. Thanks to heating in a household microwave oven the protocol of standard periodic acid–Schiff´s staining has been accelerated from 6 h to below 10 min employing standard chemistry. At the same time, we show that the microwave‐assisted glycoprotein staining is at least as sensitive as the conventional approach. All glycoproteins stained by the microwave‐accelerated procedure were successfully identified using MALDI TOF/TOF mass spectrometry. The ensuing reduction in gel staining time and simplification of the staining protocol should significantly increase laboratory throughput when glycoprotein detection on electrophoretic gels is required in large numbers.     

溴酚蓝对大鼠脑胶质瘤模型的染色作用

目的研究溴酚蓝对大鼠胶质瘤模型的染色作用，为手术精确切除人脑胶质瘤提供实验基础。方法建立Fischer 344大鼠胶质瘤模型，MRI检查证实。静脉注射不同剂量（30～360mg/kg）溴酚蓝，15min后处死取脑，检查溴酚蓝对脑肿瘤的染色情况；并注射相同剂量（180mg/kg）的溴酚蓝，在不同时间处死取脑，检查溴酚蓝对脑肿瘤的染色情况。对无瘤实验大鼠注射不同剂量溴酚蓝，观察其毒性反应。结果溴酚蓝对大鼠胶质瘤有明显的染色作用，其染色与周围脑组织有清晰的界限，染色时间出现在注射后10min～6h。30d未观察到溴酚蓝的任何毒性反应。结论溴酚蓝在不同时间点能够清晰染色大鼠胶质瘤，有可能作为人脑胶质瘤的术中染色剂来区别肿瘤组织和正常脑组织。     

STUDY ON THE MECHANISM OF STAINING BY ALLYLAMINE / OsO_4 ON PET FILM

It was proposed that in the staining process allylamine diffuses into amorphous region of PET film, reacting slightly with ester bond on PET chain and connecting with it and remaining in the amorphous area, then OsO_4 reacts with C=C group in allylamine, deposits in that region and hence, increases the contrast between amorphous and crystalline regions.     

Analysis and purification of peptide nucleic acids by denaturing PAGE

A flexible and convenient protocol for the analysis and purification of peptide nucleic acid (PNA) oligomers and PNA-peptide chimeras by denaturing PAGE is described. Vertical slab gel electrophoresis, 26% in polyacrylamide and 8 M urea at pH 3, was suitable for analysis of oligomers ranging in size from tetramers (4-mers) to tetradodecamers (24-mers). Single-base resolution of oligomers was achieved and separations are generally superior to those given by standard RP-HPLC techniques. The separation of a related series of PNA oligomers showed the distance migrated was linearly dependent on the logarithm of the molecular weight. The migration of oligomers through the gel is dependent on the number of basic functional groups present, such as amino groups, and the A and C content of the oligomer. PNAs are amenable to detection by UV-shadowing technique illuminated at 260 nm or Coomassie blue staining, both with similar, sub-microgram per band detection limits.     

A simple system for staining protein and nucleic acid electrophoresis gels

Researchers in molecular biology spend a significant amount of time tending to the staining and destaining of electrophoresis gels. Here we describe a simple system, costing approximately $100 and taking approximately 1 h to assemble, that automates standard nucleic acid and protein gel staining protocols. Staining is done in a tray or, with DNA gels, in the electrophoresis chamber itself following automatic detection of the voltage drop. Miniature pumps controlled by a microcontroller chip exchange the necessary solutions at programmed time intervals. We demonstrate efficient and highly reproducible ethidium bromide and methylene blue staining of DNA in agarose gels and Coomassie blue and silver staining of proteins in polyacrylamide gels.