细胞技术

Although immunoelectron microscopy is a powerful tool for visualizing the subcellular localization of target proteins, it is difficult to obtain and purify the specific antibodies required for this method. Instead of raising antibodies against individual target proteins, the u ...

Advances in genomic and proteomic platforms enable high-throughput studies for regulatory factors and interactors involved in signaling network at a molecular level. However, it has never been trivial to verify the omics data in vivo or functionally integrate the data in a cell signaling ...

Polyclonal antibodies are derived from multiple B-cell clones that have differentiated into antibody-producing plasma cells in response to an immunogen. Polyclonal antibodies raised against a single molecular species of antigen recognize multiple epitopes on a target molec ...

Before the advent of molecular methods to tag proteins, the visualization of proteins within cells by immunoelectron microscopy required the use of highly specific antibodies directed against the protein of interest. Thus, only proteins for which antibodies were available could be v ...

The detection of proteins with antibodies that are conjugated to gold particles has been a major asset to cell biology and the neurosciences, and knowledge about the subcellular location of antigens has formed the basis for many hypotheses regarding protein function. Many protocols have b ...

Monoclonal antibodies (mAb’s) have been used extensively in biochemical and biomedical studies, including immunoelectron microscopy. Production of mAb’s consists of four steps: immunizing the animal usually a mouse, obtaining immune cells from the spleen of the immunized mouse, f ...

Colloidal gold probes, including protein A-, IgG-F(ab’)2-, and streptavidin-labeled gold particles, are useful tools for localization of antigens in cells and tissues by immunoelectron microscopy (IEM). This chapter describes different methods for the preparation of colloidal g ...

Peptides (8–20 residues) are as effective as proteins in raising antibodies, both polyclonal and monoclonal with a titer above 20,000 easily achievable. A successful antipeptide antibody production depends on several factors such as peptide sequence selection, peptide synthesi ...

Immunoelectron microscopy is one of the best methods for detecting and localizing protein molecules in cells and tissues. Gold particles of 1.4nm in diameter (Nanogold) conjugated with Fab’ fragments easily penetrate into the cell interior and are used for pre-embedding immunoelect ...

Cryofixation and freeze-substitution techniques preserve plant ultrastructure much better than conventional chemical fixation techniques. The advantage of cryofixation is not only in structural preservation, as seen in the smooth plasma membrane, but also in the speed in arre ...

The final goal of immunohistochemical studies is that all findings examined in animal experiments should reflect the physiologically functional background. Therefore, the preservation of original components in cells and tissues is necessary for describing the functional mo ...

Mammalian tissue cultured cells are widely used in cell biology research. Immunoelectron microscopy is a powerful technique to define the subcellular localization of targeted antigens in the cultured cells. Cryofixation is now generally accepted as the best initial fixation step, ...

Immunolabelling electron microscopy is a challenging technique with demands for perfect ultrastructural and antigen preservation. High-pressure freezing offers an ideal way to fix cellular structure. However, its use for immunolabelling has remained limited because of the l ...

The freeze-fracture technique splits the frozen lipid bilayer membrane into two halves and immobilizes membrane proteins and lipids by the vacuum evaporation of platinum and carbon. After a treatment by SDS to remove extramembrane materials, the specimen is subjected to immunogold l ...

In histochemistry and cytochemistry, horseradish peroxidase-labeled lectins are often used as probes for the localization of carbohydrates in cells and tissues. In transmission electron microscopy, the most commonly used procedure for detection of carbohydrates is lectin– ...

We describe a standardized method of fixation, antigen retrieval, and image contrasting for post-embedding immunoelectron microscopy. Tissues are fixed with formaldehyde solutions containing Ca2+ and Mg2+ ions at pH 7.4 and then at pH 8.5. After dehydration with dimethylformamide, t ...

Pre-embedding nanogold silver and gold intensification methods involve immunoreactions with nanogold-labeled antibodies and intensification of the nanogold particles before embedding and ultrathin sectioning. These highly sensitive methods show good resolution ...

Multiple label immunoelectron microscopy localizes and detects multiple antigens in cells and tissues. In double labeling, two kinds of primary antibodies from different animal species are used after being mixed in a single solution. To distinguish the different antigens, seconda ...

The integral tight junction protein Claudin-16 (Cldn16) is predominantly expressed in renal epithelial cells of the thick ascending limb of Henle’s loop where, together with claudin-19, it forms a cation-selective pore that allows influx of Na+ from the interstitial fluid into the lumen of t ...

Tight junctions restrict the paracellular movement of ions, solutes, drugs, and larger material across epithelia and endothelia. For practical purposes, the barrier can be modeled as having two components. The first is a system of small 4  � radius pores lined or created by claudins. The pores show ...