treated Protocols

Category: Immunohistochemistry Protocols: Coated Treated Slides Protocols

Protocol for 3-Aminopropyltriethoxysilane treated slides. - [Read 3-Aminopropyltriethoxysilane Treated Slides Protocol]

Category: Epigenetics and Methylation Protocols: Bisulfite Treatment Methylation Assay Protocol

Yang et al, NAR. 2004. ue to the heavy methylation of repetitive elements, this assay was especially useful in detecting decreases in DNA methylation, and this assay was validated by examining cell lines treated with the methylation inhibitor 5-aza-2'deox - [Read A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements]

Category: Nucleic Acid Purification Protocols

Protocol for a single-step method for the simultaneous preparation of DNA, RNA, and protein from cells and tissues. The yield of total RNA depends on the tissue or cell source, but it is generally in the range of 4-7 µg/mg starting tissue or 5-10 µg/106 cells.  IMPORTANT: Prepare all reagents used in this protocol with Diethyl pyrocarbonate (DEPC)-treated H2O. - [Read A Single-step Method for the Simultaneous Preparation of DNA, RNA, and Protein from Cells and Tissue]

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

Method described indicates how cells are lysed by a simple freeze/thaw protocol and treated with a chemiluminescent substrate. - [Read Assay of ß-Galactosidase in Yeast: Freeze/Thaw Assay by Chemiluminescence in 96-Well Plates]

Category: Yeast Protocols: Yeast Genetics Protocols: B-Galactosidase Assay Protocols

In this protocol the cells are lysed by a simple freeze/thaw protocol and treated with a chemiluminescent substrate. - [Read Assay of ß-Galactosidase in Yeast: Freeze/Thaw Assay by Chemiluminescence in 96-Well Protocol]

Category: Neuroscience Protocols: Neuronal Stains Protocols: Stains for Senile Plaques Protocols

A silver stain to demonstrate neurofibrillary tangles, nerve fibers and senile plaques in Alzheimer's disease. The nerve fibers are sensitized with a silver solution.  The sections are treated with ammoniacal silver, and then reduced to a visible metallic silver. - [Read Bielschowsky Technique for Senile Plaques Protocol]

Category: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

Protocol for direct retrieval of DNA fragments from pulsed-field gels. A gel slice containing a fragment of DNA resolved by pulsed-field gel electrophoresis is treated with agarase.  The released DNA can be used as a substrate for ligation or restriction without further purification. - [Read Direct Retrieval of DNA Fragments from Pulsed-field Gels Protocol]

Category: RNAi Technology Protocols: RNAi Avian Embryos Protocols

Protocol describes how to dissect spinal cords of dsRNA-treated avian embryos for analysis of commissural axon guidance.  After fixation of the spinal cord with paraformaldehyde, commissural axons are stained with Fast-DiI. - [Read Dissection of Spinal Cords for Analysis of Axonal Pathfinding in dsRNA Treated Avian Embryos]

Category: Avian Bird Protocols

Protocol describes how to dissect spinal cords of dsRNA-treated avian embryos for analysis of commissural axon guidance. After fixation of the spinal cord with paraformaldehyde, commissural axons are stained with Fast-DiI. - [Read Dissection of Spinal Cords for Analysis of Axonal Pathfinding in dsRNA-Treated Avian Embryos]

Category: Cell Biology and Cell Culture: Apoptosis Protocols

DNA laddering can be detected from samples with only 8% apoptotic cells. Alternatively, the cells can be stained with DAPI and analysed by flow cytometry. CellDeath.de - [Read DNA laddering assay for treated cells]

Category: C. elegans Protocols: C. elegans Fixing Protocols

Protocol for fixation and permeabilization of C. elegans. Protocol works for all stages except dauers (which won't open) and hypoclorite-treated eggs (which disintegrate). Luckily, hypochlorite treatment and fixation are by themselves sufficient to open eggs. - [Read Fixation and Permeabilization of C. elegans Protocol]

Category: Cell Biology and Cell Culture: Cell Cycle Protocols: S Phase Protocols

This protocol provides a method for synchronizing cells at the G1/S border using a double treatment of thymidine, which, in excess, is an inhibitor of DNA synthesis. Cells are treated once with excess thymidine to accumulate the majority of them at G1/S; however, some cells will have stopped growth within the S phase. - [Read G1/S Phase Synchronization using Double Thymidine Synchronization Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Cell Cycle Analysis Protocols

This protocol provides a method for synchronizing cells at the G1/S border using a double treatment of thymidine, which, in excess, is an inhibitor of DNA synthesis. Cells are treated once with excess thymidine to accumulate the majority of them at G1/S; however, some cells will have stopped growth within the S phase. - [Read G1/S Phase Synchronization using Double Thymidine Synchronization Protocol]

Category: Cell Biology and Cell Culture: Cell Cycle Protocols: G Phase Protocols

This protocol provides a method for synchronizing cells at the G1/S border using a double treatment of thymidine, which, in excess, is an inhibitor of DNA synthesis. Cells are treated once with excess thymidine to accumulate the majority of them at G1/S; however, some cells will have stopped growth within the S phase. - [Read G1/S Phase Synchronization using Double Thymidine Synchronization Protocols]

Category: Cell Biology and Cell Culture: Cell Cycle Protocols: G Phase Protocols

This protocol uses the plant amino acid mimosine as a G1/S synchronizing agent. Cells are first treated with excess thymidine to accumulate the majority of them at G1/S; however, some cells will have stopped growth within the S phase. Thymidine is then removed to allow all the cells to proceed completely through the S phase. Mimosine is then added to arrest the cells at the G1/S border. When mimosine is removed, cells will begin to enter S phase within about 1 hour. - [Read G1/S Phase Synchronization Using Mimosine Arrest Protocol]

Category: Cell Biology and Cell Culture: Cell Cycle Protocols: S Phase Protocols

Protocol uses the plant amino acid mimosine as a G1/S synchronizing agent. Cells are first treated with excess thymidine to accumulate the majority of them at G1/S; however, some cells will have stopped growth within the S phase. Thymidine is then removed to allow all the cells to proceed completely through the S phase. Mimosine is then added to arrest the cells at the G1/S border. When mimosine is removed, cells will begin to enter S phase within about 1 hour. - [Read G1/S Phase Synchronization Using Mimosine Arrest Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Cell Cycle Analysis Protocols

This protocol uses the plant amino acid mimosine as a G1/S synchronizing agent. Cells are first treated with excess thymidine to accumulate the majority of them at G1/S; however, some cells will have stopped growth within the S phase. Thymidine is then removed to allow all the cells to proceed completely through the S phase. Mimosine is then added to arrest the cells at the G1/S border. When mimosine is removed, cells will begin to enter S phase within about 1 hour. - [Read G1/S Phase Synchronization Using Mimosine Arrest Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols: T-Cell Isolation Protocols

Protocol for isolation of T cells from synovial membranes. Includes: Initial Cell Isolation; Nonadherent Cell Isolation; Adherent Cell Isolation; Neuraminidase-Treated Sheep Red Blood Cells; Erythrocyte Lysing Solution; Balance Salt Solution (BSS). - [Read Isolation of T Cells from Synovial Membranes Protocol]

Category: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

Protocol for preparation of DNA for pulsed-field gel electrophoresis: isolation of intact DNA from yeast. Yeast cells are first treated enzymatically to break down the cell walls and then resuspended in low-melting-temperature agarose plugs.  The DNA is liberated by infusing the plugs with lysis buffer and proteases.  This method is used to prepare both conventional and artificial yeast chromosomes. - [Read Preparation of DNA for Pulsed-field Gel Electrophoresis: Isolation of Intact DNA from Yeast]

Category: Stem Cell Protocols: Feeder Cell Preparation Protocols

MEF feeders are prepared weekly to provide a substrate for undifferentiated embryonic stem (ES) cells. Primary MEF cells are thawed, established in culture, treated with mitomycin C to halt their proliferation so they cannot overgrow the ES cultures, and then replated onto dishes convenient for ES cell culture. This protocol can also be used to prepare feeder cells from STO fibroblast cell lines. - [Read Preparation of Mouse Embryonic Fibroblast (MEF) Feeder Plates Protocol]

Category: Fungi Protocols

Protocol for the preparation of solid tissue for Aspergillus galactomannan antigen detection by Platelia (Biorad). Technique was designed for use on human serum. However, it may also be possible to perform this method on solid tissues and organic solutions. Viscous solution and tissue specimens need to be pre-treated to achieve the extraction of the Aspergillus antigen and to get a homogeneous sample in solution. - [Read Preparation of Solid Tissue for Aspergillus Galactomannan Antigen Detection by Platelia Protocol]

Category: Drosophila Protocols: Drosophila Staining Protocols

Early embryos (0-17 hours or until cuticle formation) are treated with a mixture of organic solvents, formaldehyde, and alcohols, as described here. The cuticles of late-stage embryos are usually opened by sonication. Tissues from more advanced stages of development are normally dissected by hand and then fixed and stained in a standard paraformaldehyde/detergent combination - [Read Preparing Early Whole-Mount Drosophila Embryos for Immunostaining Protocol]

Category: Drosophila Protocols: Drosophila Staining Protocols

Early and late embryos are treated with a mixture of organic solvents, formaldehyde, and alcohols. The cuticles of late-stage embryos (17-22 hours or until hatching) are usually opened by sonication, as described here. Tissues from later stages of development are normally dissected by hand and then fixed and stained in a standard paraformaldehyde/detergent combination. - [Read Preparing Late Whole-Mount Drosophila Embryos for Immunostaining Protocol]

Category: Microscopy Protocols: In Vivo Imaging Protocols

To image early cleavages and chromatin dynamics, it is convenient to use histone H2B fused to GFP or lamin::GFP. Time-lapse movies can be obtained using conventional confocal microscope systems and their included software. Early embryos dissected from transgenic hermaphrodites are placed with egg salts on agar pads. Chromatin dynamics can be followed easily, and wild-type embryonic cells can be compared with mutants or RNAi-treated embryos. - [Read Protocol Live Imaging of Caenorhabditis Elegans]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Crude preparations of plasmid DNA are first treated with lithium chloride and Rnase (to remove RNA).  The plasmid DNA is then precipitated in a solution containing polyethylene glycol and MgCl2. - [Read Purification of Plasmid DNA by Precipitation with Polyethylene Glycol Protocol]