3,206 Works

Cutaneous Two-Stage Chemical Carcinogenesis

Sprotocols
Authors: Renata B. Filler, Scott J. Roberts and Michael Girardi Corresponding author ([michael.girardi@yale.edu](michael.girardi@yale.edu)) ### INTRODUCTION The induction of cutaneous carcinogenesis in mice by topical administration of chemicals enables the local, systemic, and environmental factors that influence tumor susceptibility, growth, and progression to be studied in the laboratory. Chemical carcinogenesis in mouse skin has been used for several decades, and continues to help in the identification of important molecular and immunological pathways involved in cutaneous malignancy....

Sequential Rna And Dna Fluorescence In Situ Hybridization

Sprotocols
Authors: Takumi Takizawa and Tom Misteli ### Introduction An increasing body of evidence indicates that the spatial positioning of genes in the interphase nucleus is highly relevant for their function (Lanctot et al, 2007; Meaburn & Misteli, 2007; Misteli, 2007). Fluorescence in situ hybridization (FISH) is a powerful technique to map gene loci in the interphase nucleus. Depending on protocol FISH can either detect DNA or RNA. Both methods have limitations. DNA FISH only detects...

Tissue Preparation For In Situ Hybridization

Sprotocols
Author: Josiah N. Wilcox 1. Harvest tissue and rinse in PBS or saline. - Immerse tissue in 4% paraformaldehyde/0.1M sodium phosphate buffer pH7.4 (recipe follows) at 4°C for 1-3hrs. Try to avoid overnight fixation if possible as this causes problems with tissue adherence on slides during the hybridization procedure. - Immerse tissue in sterile 15% sucrose/1xPBS (recipe follows) 3 hrs. to overnight at 4°C. - Embed tissue in O.C.T. (Baxter No. M7148-4), M1 (Lipshaw) or...

Colony Screening By Pcr

Sprotocols
Author: Matt Lewis ### Notes This is the fastest way to screen bacterial colonies. Our PCR machine takes 24 tubes so I routinely screen 22 colonies + 1 negative + 1 positive control. ### Choosing the primers Ideally you want a primer pair that can only work if the correct construct is present eg. a vector flanking primer and a gene specific primer. However, this may not allow you a positive control (essential) so you...

Rna In Situ Protocol For Dna And Rna Probes

Sprotocols
Author: Grace Panganiban This procedure was adapted from one originally described by Tautz and Pfeifle (1989). It makes use of digoxigenin labelled probes and alkaline phosphatase-conjugated anti-digoxigenin antibody fragments (Boehringer Mannheim Biochemicals) to detect the hybridizing RNA in whole embryos. The major modification made was to increase the number of post-hybridization washes in hybridization buffer (50% deionized formamide, 5x SSC, 200 ug/ml tRNA, 100 ug/ml sonicated, boiled salmon sperm DNA, 0.1% Tween-20) and the duration...

Hiro Hirai'S Bac-Fish Protocol

Sprotocols
Author: Schistosoma Genome Network ### Overview This protocol is a modification of the standard FISH protocol published by Hiro Hirai and Phil LoVerde in Parasitology Today (1995, 11(8) p 310-314) that has been optimised for use with BAC probes and should be read in conjunction with the published protocol. This protocol uses the BIOPRIME reaction kit from GibcoBRL to prepare biotin-labelled BAC DNA which is detected using FITC-Avidin (Vector Labs, DCS grade). Reagents from other...

Protein-Dna Telomere Fish

Sprotocols
Author: Stewart Laboratory ### FIX AND PROTEIN STRAIN 1. Fix cells in 4% paraformaldhyde (diluted in PBS) for 10min at RT - Wash cells with PBS +0.2% Tween20 - Permeabilize cells with 0.5% Triton X-100 for 10 min at RT - Wash cells with PBS +0.2% Tween20 - Block cells with blocking buffer for 15 min at 37°C (We use 0.2% Tween20 and 10% serum) - Stain cells with antibody of interest diluted in the...

Hiro Hirai'S Bac-Fish Protocol

Sprotocols
Author: Schistosoma Genome Network ### Overview This protocol is a modification of the standard FISH protocol published by Hiro Hirai and Phil LoVerde in Parasitology Today (1995, 11(8) p 310-314) that has been optimised for use with BAC probes and should be read in conjunction with the published protocol. This protocol uses the BIOPRIME reaction kit from GibcoBRL to prepare biotin-labelled BAC DNA which is detected using FITC-Avidin (Vector Labs, DCS grade). Reagents from other...

Autoradiography

Sprotocols
Author: Dr. William H. Heidcamp ### Materials 1. ^3H-Thymidine, specific activity of 2.0 - curie/millimole - Onion sets, jars and toothpicks - Alcohol-acetic acid fixative - Materials for feulgen reaction (Exercise 2.4) - Paraffin embedding, sectioning equipment - Kodak Nuclear Track Emulsion (or equivalent) - Darkroom - Water bath at 42° C - Kodak D19 Developer - Kodak Fixer - Xylol, Permount and coverslips - Microscope ### Procedure 1. Carefully read and follow all precautions...

Brown Fat Cell Isolation

Sprotocols
Author: C.R. Kahn ### 1.) ISOLATION AND PRIMARY CULTURE OF BROWN FAT PREADIPOCYTES ### Rationale: To prepare primary brown preadipocytes for immortalization: useful for metabolic studies from knockout mice. This consists of the following five protocols. References: Fasshauer, M., J. Klein, K M. Kriauciunas, K. Ueki, M.Benito, and C.R. Kahn. 2001. Essential role of insulin substrate 1 in differentiation of brown adipocytes. *Mol Cell Biol* 21: 319-329. Fasshauer, M., J. Klein, K. Ueki, K.M. Kriauciunas,...

In Situ Hybridisation To Alpha Satellite Sequences (Chromosome Specific)

Sprotocols
Author: Matt Lewis ### Notes Alpha satellite sequences, whilst highly repetitive, are specific to each individual chromosome. These sequences flank the centromeres and can present a target measured in megabases. In this protocol a biotin or digoxigenin labelled DNA probe is detected using HRP-conjugated antibodies. The signal is visualised with diaminobenzidine (DAB). Normal, healthy nuclei show two spots. Aneusomic nuclei show 1, 3, 4 or more spots. ![Figure 1](https://i.imgur.com/to3jB0j.jpg "Figure 1") **Figure 1**. A 6mm...

Assay For Free Fatty Acid Release From Isolated Fat Cells

Sprotocols
Author: Michael P. Czech This assay is used to measure free fatty acid release from fat cells as an estimate of lipolysis. Glycerol release can also be measured from the same samples by taking an aliquot of medium from below the fat cell layer and assaying for glycerol in the medium. *Note: This protocol contains dangerous chemicals that can cause burns to exposed skin and should not be inhaled; take all precautions to prevent burns...

96-Well Rna In Situ Hybridization Protocol

Sprotocols
Author: Berkeley Drosophila Genome Project ### 1. Materials **1.1. Probe Preparation** **1.1.1. Cell Inoculation** 1. 96-well, deep square-well round bottom plate (E&K; Scientific Ritter Riplate). - Super Broth (SB: 10 gm 4-Morpholinepropanesulfonic acid (MOPS), 20 gm Bacto Yeast Extract, 30 gm Tryptone Peptone/L). - Antibiotic (80 µg/ml final concentration Carbenicillin- for pFLC-I or pBS SK, 80 µg/ml chloramphenicol for pOT2 or pOTB7). - Multichannel pipette (Brand). - Multichannel pipette (Qiagen Multi Electrapette 1550). - Airpore...

Adult Cattle Serum As Supplemental Nutrient In Lymphocyte Cultures For Chromosomes Studies

Sprotocols
Author: B. R. Yadav ### Abstract: The author has made a search for an alternate nutrient (serum supplement) source for lymphocyte cultures. This report describes the procedure for separation of serum from adult cattle and its successful use, which gives an excellent mitotic index for chromosomes investigations. This procedure can be applied practically in all the laboratories requiring serum, particularly in the countries with limitations on cow slaughtering. In this process there is no need...

Adult Cattle Serum As Supplemental Nutrient In Lymphocyte Cultures For Chromosomes Studies

Sprotocols
Author: B. R. Yadav ### Abstract: The author has made a search for an alternate nutrient (serum supplement) source for lymphocyte cultures. This report describes the procedure for separation of serum from adult cattle and its successful use, which gives an excellent mitotic index for chromosomes investigations. This procedure can be applied practically in all the laboratories requiring serum, particularly in the countries with limitations on cow slaughtering. In this process there is no need...

Clonality - X Chromosome Inactivation Assay

Sprotocols
Author: Molecular Profiling Initiative, NCI This method was successful in our lab using prostate tissue and for our specific objectives. Investigators must be aware that they will need to tailor the following protocol for their own research objectives and tissue under study. Investigators can utilize X chromosome inactivation (methylation) to determine the clonality status of a tumor or premalignant lesion in females. The technique is based on a methylation-sensitive restriction enzyme and analysis of a...

Adult Cattle Serum As Supplemental Nutrient In Lymphocyte Cultures For Chromosomes Studies

Sprotocols
Author: B. R. Yadav ### Abstract: The author has made a search for an alternate nutrient (serum supplement) source for lymphocyte cultures. This report describes the procedure for separation of serum from adult cattle and its successful use, which gives an excellent mitotic index for chromosomes investigations. This procedure can be applied practically in all the laboratories requiring serum, particularly in the countries with limitations on cow slaughtering. In this process there is no need...

Chromosome Conformation Capture

Sprotocols
Author: Marcus Marvin ### Nuclei preparation **Preparing the cells** 1. 300–500 ml cultures of density 1.5–4.0 x 10e7 cells/ml - Fix cells with freshly prepared formaldehyde in Buffer A for 2 minutes, stirring, to a final concentration of 1% - Quench fixation by adding glycine to final concentration of 0.125 M - Wash cells twice in Buffer A - Resuspend cells in 5 ml Spheroplasting Buffer and check for lysis **Extract nuclei as follows (taken...

Loss Of Heterozygosity

Sprotocols
Author: Molecular Profiling Initiative, NCI *This method was successful in our lab using prostate tissue and for our specific objectives. Investigators must be aware that they will need to tailor the following protocol for their own research objectives and tissue under study*. This method is used to detect genomic DNA deletions in tumor cells. For a more detailed discussion of applying this approach to microdissected samples, see [Allelic Loss Studies](http://cgap-mf.nih.gov/ProstateExample/ProstateMAandResults/AllelicLossStudies.html) in [Prostate MP at NCI](http://cgap-mf.nih.gov/ProstateExample/index.html)....

Adhesive Micropatterns For Cells: A Microcontact Printing Protocol

Sprotocols
Authors: Manuel Théry and Matthieu Piel Corresponding authors ([manuel.thery@cea.fr](manuel.thery@cea.fr); [matthieu.piel@curie.fr](matthieu.piel@curie.fr)) ### INTRODUCTION This protocol describes a simple, fast, and efficient method for making adhesive micropatterns that can be used to control individual cell shape and adhesion patterns. It is based on the use of an elastomeric stamp containing microfeatures to print proteins on the substrate of choice. The process can be subdivided into three parts. First, a silicon master is fabricated, which contains the microfeatures...

Cultures Of Cerebellar Granule Neurons

Sprotocols
Authors: Parizad M. Bilimoria and Azad Bonni1 Corresponding author ([azad_bonni@hms.harvard.edu](azad_bonni@hms.harvard.edu)) ### INTRODUCTION Primary cultures of granule neurons from the post-natal rat cerebellum provide an excellent model system for molecular and cell biological studies of neuronal development and function. The cerebellar cortex, with its highly organized structure and few neuronal subtypes, offers a well-characterized neural circuitry. Many fundamental insights into the processes of neuronal apoptosis, migration, and differentiation in the mammalian central nervous system have come...

Calcium Imaging Of Neuronal Circuits In Vivo Using A Circuit-Tracing Pseudorabies Virus

Sprotocols
Authors: Andrea E. Granstedt, Bernd Kuhn, Samuel S.-H. Wang and Lynn W. Enquist Corresponding author ([lenquist@princeton.edu](lenquist@princeton.edu)). ### INTRODUCTION Pseudorabies virus (PRV) is a neuroinvasive virus of the herpes family that has a broad host range but does not infect higher-order primates. PRV characteristically travels along chains of synaptically connected neurons and has been used extensively for elucidating neural circuits in the peripheral and central nervous system in vivo. The recombinant virus PRV369 is an attenuated...

Array Tomography: High-Resolution Three-Dimensional Immunofluorescence

Sprotocols
Authors: Kristina D. Micheva, Nancy O’Rourke, Brad Busse and Stephen J. Smith Adapted from [*Imaging: A Laboratory Manual* ](http://www.cshlpress.com/link/imagingp.htm)(ed. Yuste). CSHL Press, Cold Spring Harbor, NY, USA, 2010. ### INTRODUCTION Array tomography, which is described in this article, is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting...

Array Tomography: High-Resolution Three-Dimensional Immunofluorescence

Sprotocols
Authors: Kristina D. Micheva, Nancy O’Rourke, Brad Busse and Stephen J. Smith Adapted from [*Imaging: A Laboratory Manual* ](http://www.cshlpress.com/link/imagingp.htm)(ed. Yuste). CSHL Press, Cold Spring Harbor, NY, USA, 2010. ### INTRODUCTION Array tomography, which is described in this article, is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting...

Combined Flow Cytometric Measurement Of Two Cell-Surface Antigens And Dna-Rna Content

Sprotocols
Author: Ingrid Schmid Corresponding author ([schmid@mednet.ucla.edu](schmid@mednet.ucla.edu)) ### INTRODUCTION Flow cytometry is frequently used to assess nucleic acid content in individual cells. Based on DNA content alone, however, cells in the quiescent G0 phase cannot be discriminated from cells in the proliferative G1 phase, as DNA content remains constant until S-phase entry. In contrast, by measuring RNA content in addition to DNA content, cells can be assigned to G0 and cell-cycle subcompartments of G1. Assessing phenotype...

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