Indirect immunofluorescence with preextraction (in situ cell fractionation)

2006 ◽  
Author(s):  
Raffaella Di Micco ◽  
Fabrizio d'Adda di Fagagna
Keyword(s):  
Indirect Immunofluorescence ◽  
Cell Fractionation

Multiple Iteractive Labeling by Antibody Neodeposition (MILAN)

2019 ◽  
Author(s):  
Giorgio Cattoretti ◽  
Francesca Maria Bosisio ◽  
Lukas Marcelis ◽  
Maddalena Maria Bolognesi
Keyword(s):  
Image Analysis ◽  
Tissue Section ◽  
Indirect Immunofluorescence ◽  
Analysis Software ◽  
Tissue Sections ◽  
Image Analysis Software ◽  
Single Section ◽  
Fluorescent Image ◽  
Secondary Antibodies

Abstract Multiplexing, labeling for multiple immunostains the very same cell or tissue section in situ, is of considerable interest. The major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, scarcity of specialized skills or facilities. We have validated and detail here a method based on common primary and secondary antibodies, diffusely available fluorescent image scanners and routinely processed tissue sections \(FFPE). It entails rounds of four-color indirect immunofluorescence, image acquisition and removal \(stripping) of the antibodies, before another stain is applied. The images are digitally registered and the autofluorescence is subtracted. Removal of antibodies is accomplished by disulphide cleavage. In excess of 50 different antibody stains can be applied to one single section from routinely fixed and embedded tissue. This method requires a modest investment in hardware and materials and uses freeware image analysis software.

scholarly journals Chinese hamster ovary cell lysosomes retain pinocytized horseradish peroxidase and in situ-radioiodinated proteins.

1984 ◽  
Vol 4 (2) ◽  
pp. 296-301 ◽  
Author(s):  
B Storrie ◽  
M Sachdeva ◽  
V S Viers
Keyword(s):  
Horseradish Peroxidase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fluid Phase ◽  
Ovary Cell ◽  
Cell Fractionation ◽  
Marker Enzyme ◽  
Ovary Cells

We used Chinese hamster ovary cells, a cell line of fibroblastic origin, to investigate whether lysosomes are an exocytic compartment. To label lysosomal contents, Chinese hamster ovary cells were incubated with the solute marker horseradish peroxidase. After an 18-h uptake period, horseradish peroxidase was found in lysosomes by cell fractionation in Percoll gradients and by electron microscope cytochemistry. Over a 24-h period, lysosomal horseradish peroxidase was quantitatively retained by Chinese hamster ovary cells and inactivated with a t 1/2 of 6 to 8 h. Lysosomes were radioiodinated in situ by soluble lactoperoxidase internalized over an 18-h uptake period. About 70% of the radioiodine incorporation was pelleted at 100,000 X g under conditions in which greater than 80% of the lysosomal marker enzyme beta-hexosaminidase was released into the supernatant. By one-dimensional electrophoresis, about 18 protein species were present in the lysosomal membrane fraction, with radioiodine incorporation being most pronounced into species of 70,000 to 75,000 daltons. After a 30-min or 2-h chase at 37 degrees C, radioiodine that was incorporated into lysosomal membranes and contents was retained in lysosomes. These observations indicate that lysosomes labeled by fluid-phase pinocytosis are a terminal component of endocytic pathways in fibroblasts.

Chemical components and their locations in the Verticillium fungicola cell wall

2000 ◽  
Vol 46 (2) ◽  
pp. 101-109 ◽  
Author(s):  
M Calonje ◽  
M Novaes-Ledieu ◽  
D Bernardo ◽  
O Ahrazem ◽  
C García Mendoza
Keyword(s):  
Indirect Immunofluorescence ◽  
Cell Walls ◽  
Agaricus Bisporus ◽  
Chemical Structure ◽  
Wall Material ◽  
Chemical Components ◽  
Carbohydrate Polymers ◽  
Verticillium Fungicola ◽  
Intermediate Layers

The chemical structure of cell walls and fractions of Verticillium fungicola, a pathogen of Agaricus bisporus, as well as their corresponding ultrastructures were studied. There are at least three chemically distinct types of carbohydrate polymers: one yielding mannose with lower amounts of galactose and glucose (glucogalactomannan), another one composed mainly of glucose (glucan), and a third one containing only N-acetylglucosamine (chitin). Attempts were made to locate these materials in situ by comparing electron micrographs of shadowed and sectioned cell walls, and also by indirect immunofluorescence. It was shown that none of these polymers constituted a completely physically distinct layer, but there seem to be different solubility properties in the outer, inner, and intermediate layers. It was also shown that fibrillar material (chitin) embedded in cementing glucan constituted the residual inner fraction of the original wall material. Indirect immunofluorescence showed the location of a significant amount of glucogalactomannan on the surface of the walls in which rodlet structures were visualized by electron microscopy.Key words: cell walls, polysaccharides, Verticillium fungicola.

scholarly journals Simple diagnostic test for antibodies to Encephalitozoon cuniculi based on enzyme immunoassay

1981 ◽  
Vol 15 (1) ◽  
pp. 41-44 ◽  
Author(s):  
J. C. Cox ◽  
R. Horsburgh ◽  
D. Pye
Keyword(s):  
Cell Culture ◽  
Enzyme Immunoassay ◽  
Diagnostic Test ◽  
Indirect Immunofluorescence ◽  
Antigen Processing ◽  
Encephalitozoon Cuniculi ◽  
Immunofluorescence Test ◽  
Indirect Immunofluorescence Test ◽  
Specialized Equipment

Rabbit antibodies against Encephalitozoon cuniculi were detected in an enzyme immunoassay procedure in which antigen was grown and used in situ. The test appeared to be more sensitive than the indirect immunofluorescence test with which it was compared, but gave essentially the same results for the 64 sera evaluated. This procedure will allow any laboratory with cell-culture facilities to produce a diagnostic antigen without the need for antigen processing. It is simple and reliable, and does not require specialized equipment or microscopic assessment.

scholarly journals A small RNA in testis and brain: implications for male germ cell development

2002 ◽  
Vol 115 (6) ◽  
pp. 1243-1250
Author(s):  
Ilham A. Muslimov ◽  
Yuan Lin ◽  
Michal Heller ◽  
Jürgen Brosius ◽  
Zahra Zakeri ◽  
...  
Keyword(s):  
Germ Cells ◽  
Translational Control ◽  
Rna Polymerase Iii ◽  
Cell Fractionation ◽  
Gene Encoding ◽  
Male Germ Cells ◽  
Local Protein Synthesis ◽  
Non Coding Rna ◽  
Developmental Analysis

BC1 RNA, a small non-coding RNA polymerase III transcript, is selectively targeted to dendritic domains of a subset of neurons in the rodent nervous system. It has been implicated in the regulation of local protein synthesis in postsynaptic microdomains. The gene encoding BC1 RNA has been suggested to be a master gene for repetitive ID elements that are found interspersed throughout rodent genomes. A prerequisite for the generation of repetitive elements through retroposition and subsequent transmission in the germline is expression of the master gene RNA in germ cells. To test this hypothesis, we have investigated expression of BC1 RNA in murine male germ cells. We report that BC1 RNA is expressed at substantial levels in a subset of male germ cells. Results from cell fractionation experiments, developmental analysis,and northern and in situ hybridization showed that the RNA was expressed in pre-meiotic spermatogonia, with particularly high amounts in syncytial ensembles of cells that are primed for synchronous spermatogenic differentiation. BC1 RNA continued to be expressed in spermatocytes, but expression levels decreased during further spermatogenic development, and low or negligible amounts of BC1 RNA were identified in round and elongating spermatids. The combined data indicate that BC1 RNA operates in groups of interconnected germ cells, including spermatogonia, where it may function in the mediation of translational control. At the same time, the identification of BC1 RNA in germ cells provides essential support for the hypothesis that repetitive ID elements in rodent genomes arose from the BC1 RNA gene through retroposition.

scholarly journals Detection of Streptococcus Suis by in Situ Hybridization, Indirect Immunofluorescence, and Peroxidase-Antiperoxidase Assays in Formalin-Fixed, Paraffin-Embedded Tissue Sections from Pigs

2000 ◽  
Vol 12 (3) ◽  
pp. 224-232 ◽  
Author(s):  
Mette Boye ◽  
Anne A. Feenstra ◽  
Conny Tegtmeier ◽  
Lars Ole Andresen ◽  
Søren R. Rasmussen ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Indirect Immunofluorescence ◽  
Polyclonal Antibodies ◽  
Single Cells ◽  
Streptococcus Suis ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Streptococcus suis is an important pathogen in pigs and is considered a zoonotic agent. To aid diagnosis of infection caused by S. suis, a species-specific probe targeting 16S ribosomal RNA was designed and used for fluorescent in situ hybridization. Two additional immunohistochemical detection methods, an indirect immunofluorescence assay and a peroxidase-antiperoxidase method, using polyclonal antibodies also were developed. The specificity of the oligonucleotide probe was examined by whole-cell and dot-blot hybridization against reference strains of the 35 serotypes of S. suis and other closely related streptococci and other bacteria commonly isolated from pigs. The probe was specific for S. suis serotypes 1–31. The specificity of the polyclonal antibodies, which has previously been evaluated for use in diagnostic bacteriology for typing of serotype 2, was further evaluated in experimentally infected murine tissue with pure culture of different serotypes of S. suis, related streptococci, and other bacteria commonly found in pigs. The polyclonal antibodies against S. suis serotype 2 cross-reacted with serotypes 1 and 1/2 in these assays. The in situ hybridization and the immunohistochemical methods were used for detection of S. suis in formalin-fixed, paraffin-embedded tissue sections of brain, endocardium, and lung from pigs infected with S. suis. The methods developed were able to detect single cells of S. suis in situ in the respective samples, whereas no signal was observed from control tissue sections that contained organisms other than S. suis. These techniques are suitable for determining the in vivo localization of S. suis for research and diagnostic purposes.

scholarly journals Characterization of a rapid cellular-fractionation technique for hepatocytes. Application in the measurement of mitochondrial membrane potential in situ

1984 ◽  
Vol 219 (2) ◽  
pp. 375-382 ◽  
Author(s):  
S B Shears ◽  
C J Kirk
Keyword(s):  
Incubation Medium ◽  
Plasma Membranes ◽  
Electrical Potential ◽  
Cell Fractionation ◽  
Marker Enzyme ◽  
Cell Nuclei ◽  
Electrical Potentials

A rapid cellular-fractionation technique [Hoek, Nicholls & Williamson (1980) J. Biol. Chem. 255, 1458-1464] was further characterized by using hepatocytes. Of the mitochondrial marker-enzyme activity, 80% was routinely separated from 71-98% of the total cell activities of marker enzymes for plasma membranes, Golgi-membranes, endoplasmic reticulum, lysosomes and cytosol. The mitochondria were contaminated with 53% of cell nuclei. [3H]Triphenylmethylphosphonium ion (TPMP+) was added to hepatocytes in an attempt to measure cellular transmembrane electrical potentials. After rapid cell fractionation the electrical potential between mitochondria in situ and the incubation medium was found to be 202 mV. This value was slightly increased when hepatocytes were treated with oligomycin, but substantially decreased by oligomycin plus an uncoupler of oxidative phosphorylation. Although estimates of TPMP+ binding were obtained, substantial difficulties prevented the accurate measurement of the electrical potential across the plasma membrane. It is concluded that TPMP+ may be employed to demonstrate the integrity of mitochondria during the fractionation procedures. However, the cation is inadequate for the determination of the separate components of the electrical potential between the mitochondrial matrix and the incubation medium.

scholarly journals Preparation of nuclear matrices from cultured cells: subfractionation of nuclei in situ.

1984 ◽  
Vol 98 (5) ◽  
pp. 1886-1894 ◽  
Author(s):  
M Staufenbiel ◽  
W Deppert
Keyword(s):  
Cultured Cells ◽  
Cellular Structures ◽  
Structural Systems ◽  
Cell Fractionation ◽  
Cross Contamination ◽  
Extraction Step ◽  
Before And After ◽  
Cytoskeletal Elements

Analyses of the different structural systems of the nucleus and the proteins associated with them pose many problems. Because these systems are largely overlapping, in situ localization studies that preserve the in vivo location of proteins and cellular structures often are not satisfactory. In contrast, biochemical cell fractionation may provide artifactual results due to cross-contamination of extracts and structures. To overcome these problems, we have developed a method that combines biochemical cell fractionation and in situ localization and leads to the preparation of a residual cellular skeleton (nuclear matrix and cytoskeletal elements) from cultured cells. This method's main feature is that cell fractionation is performed in situ. Therefore, structures not solubilized in a particular extraction step remain attached to the substrate and retain their morphology. Before and after each extraction step they can be analyzed for the presence and location of the protein under study by using immunological or cytochemical techniques. Thereby the in vivo origin of a protein solubilized in a particular extraction step is determined. The solubilized protein then may be further characterized biochemically. In addition, to allow analyses of proteins associated with the residual cellular skeleton, we have developed conditions for its solubilization that do not interfere with enzymatic and immunological studies.

In situ localization of chalcone synthase inLarix needles by indirect immunofluorescence

PROTOPLASMA ◽  
1989 ◽  
Vol 153 (1-2) ◽  
pp. 58-61 ◽  
Author(s):  
A. Lembach ◽  
L. Beerhues ◽  
R. Wiermann
Keyword(s):  
Indirect Immunofluorescence ◽  
Chalcone Synthase
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