Double Labeling Fluorescent Immunocytochemistry

2021 ◽  
pp. 147-161
Author(s):  
Monika Rak ◽  
Krzysztof Reiss
Keyword(s):  
Double Labeling

Gold labeling of teichoic acid on adjacent surfaces of staphylococcus aureus

1990 ◽  
Vol 48 (3) ◽  
pp. 578-579
Author(s):  
Margaret Hukee
Keyword(s):  
Staphylococcus Aureus ◽  
Teichoic Acid ◽  
Protein Labeling ◽  
Minimal Amount ◽  
Section Thickness ◽  
Double Labeling ◽  
Parallel Surfaces ◽  
Supporting Membrane

Gold labeling of two antigens (double labeling) is often done on two section surfaces separated by section thickness. Whether labeling is done on both sides of the same section or on two parallel surfaces separated by section thickness (PSSST), comparable results are dependent on an equal number of epitopes being exposed at each surface. We propose a method to study protein labeling within the same field of proteins, by examining two directly adjacent surfaces that were split during sectioning. The number of labeling sites on adjacent surfaces (AS) were compared to sites on PSSST surfaces in individual bacteria.Since each bacteria needed to be recognizable in all three section surfaces, one-hole grids were used for labeling. One-hole grids require a supporting membrane and excessive handling during labeling often ruptures the membrane. To minimize handling, a labeling chamber was designed that is inexpensive, disposable, minimizes contamination, and uses a minimal amount of solution.

Expression and distribution of the Na+- HCO 3 − cotransporter in human pancreas

1999 ◽  
Vol 277 (2) ◽  
pp. G487-G494 ◽  
Author(s):  
Christopher R. Marino ◽  
Virginia Jeanes ◽  
Walter F. Boron ◽  
Bernhard M. Schmitt
Keyword(s):  
Northern Blot ◽  
Islet Cells ◽  
Rat Kidney ◽  
Physiological Role ◽  
Human Pancreas ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cellular Mechanisms ◽  
Double Labeling ◽  
Normal Human

The cellular mechanisms of [Formula: see text] secretion in the human pancreas are unclear. Expression of a Na+-[Formula: see text]cotransporter (NBC) mRNA has been observed recently, but the distribution and physiological role of the NBC protein are not known. Here we examined the expression and localization of NBC in human pancreas by Northern blot, immunoblot, and immunofluorescence microscopy. Rat kidney NBC probes detected a single 9.5-kb band by Northern blot. On immunoblots, two polyclonal antisera directed against different epitopes of rat kidney NBC identified a single ∼130-kDa protein. In cryosections of normal human pancreas, both antisera labeled basolateral membranes of large, morphologically identifiable ducts and produced a distinct labeling pattern in the remainder of the parenchyma. In double-labeling experiments, NBC immunoreactivity in the parenchyma colocalized with the Na+-K+pump, a basolateral marker. In contrast, NBC and cystic fibrosis transmembrane conductance regulator, an apical membrane marker, were detected within the same histological structures but at different subcellular localizations. The NBC antisera did not label acinar or islet cells. Our observations suggest that secretion of[Formula: see text] by human pancreatic duct cells involves the basolateral uptake of Na+and[Formula: see text] via NBC, an electrogenic Na+-[Formula: see text]cotransporter.

scholarly journals Secretogranin II binds to secretogranin III and forms secretory granules with orexin, neuropeptide Y, and POMC

2009 ◽  
Vol 202 (1) ◽  
pp. 111-121 ◽  
Author(s):  
Kikuko Hotta ◽  
Masahiro Hosaka ◽  
Atsushi Tanabe ◽  
Toshiyuki Takeuchi
Keyword(s):  
Body Weight ◽  
Neuropeptide Y ◽  
Secretory Granules ◽  
Immunohistochemical Analysis ◽  
Mrna Levels ◽  
Weight Changes ◽  
Body Weight Changes ◽  
Hypothalamic Area ◽  
Double Labeling ◽  
Secretogranin Ii

Functional variations in the secretogranin III (SCG3) gene are associated with susceptibility to obesity. SCG3 forms secretory granules with orexin, melanin-concentrating hormone (MCH), neuropeptide Y (NPY), and POMC in the hypothalamus. In this study, we screened proteins for SCG3-binding activity and identified secretogranin II (SCG2) using a yeast two-hybrid system. Immunoprecipitation revealed that SCG2 interacts with SCG3. In situ hybridization and immunohistochemistry indicated that SCG2 was highly expressed in the lateral hypothalamic area, paraventricular nucleus, and arcuate nucleus of the hypothalamus. Double-labeling immunohistochemical analysis demonstrated that SCG2 was expressed in orexin-, MCH-, NPY-, and POMC-expressing neurons. SCG2 was also coexpressed with SCG3. Upon introduction into neuroblastoma cells, SCG2 was expressed in the cytosol and formed granule-like structures with SCG3, orexin, NPY, or POMC. SCG3 bound to POMC; however, it did not bind to orexin, MCH, or NPY. By contrast, SCG2 formed aggregates with orexin, MCH, NPY, and POMC. SCG2 may act as a hormone carrier for orexin, MCH, NPY, and POMC by binding with SCG3, which targets proteins to the secretory granules. SCG2 mRNA levels increased along with those of SCG3, orexin, MCH, and NPY after a 24-h fast, suggesting that the SCG2/SCG3 system may respond in an adaptive manner to acute body weight changes. However, this SCG2/SCG3 system appears to be unresponsive to chronic body weight changes, such as diet-induced obesity or obesity in ob/ob mice. We suggest that SCG2, as well as SCG3, may be a potential regulator of food intake based on its capacity to accumulate appetite-related hormones into secretory granules.

scholarly journals Ultrastructure of the yeast actin cytoskeleton and its association with the plasma membrane.

1994 ◽  
Vol 125 (2) ◽  
pp. 381-391 ◽  
Author(s):  
J Mulholland ◽  
D Preuss ◽  
A Moon ◽  
A Wong ◽  
D Drubin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Binding Proteins ◽  
Ultrastructural Level ◽  
Actin Binding ◽  
Cortical Actin ◽  
Actin Binding Proteins ◽  
Double Labeling ◽  
Wall Growth ◽  
Cortical Cytoskeleton

We characterized the yeast actin cytoskeleton at the ultrastructural level using immunoelectron microscopy. Anti-actin antibodies primarily labeled dense, patchlike cortical structures and cytoplasmic cables. This localization recapitulates results obtained with immunofluorescence light microscopy, but at much higher resolution. Immuno-EM double-labeling experiments were conducted with antibodies to actin together with antibodies to the actin binding proteins Abp1p and cofilin. As expected from immunofluorescence experiments, Abp1p, cofilin, and actin colocalized in immuno-EM to the dense patchlike structures but not to the cables. In this way, we can unambiguously identify the patches as the cortical actin cytoskeleton. The cortical actin patches were observed to be associated with the cell surface via an invagination of plasma membrane. This novel cortical cytoskeleton-plasma membrane interface appears to consist of a fingerlike invagination of plasma membrane around which actin filaments and actin binding proteins are organized. We propose a possible role for this unique cortical structure in wall growth and osmotic regulation.

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