Multicolour3DFISHinvertebratecells6
Back to topReviewer CommentsReviewed by: Luis Antonio Parada, CIC Biogune, Derio, Spain.In my experience the primers are better preserved when kept at higher concentration (100µM) and different volumes are added according to the required concentrations. Usually the amplification products (unlabeled) are purified by precipitation with ethanol and dissolved in bi-distilled water at high concentration to preserve the DN......阅读全文
Multicolour－3DFISH－in－vertebrate－cells1
IntroductionMulticolour 3D-FISH in combination with confocal microscopy, 3D image reconstruction and quantitative image analysis is an efficient tool
Multicolour－3DFISH－in－vertebrate－cells6
Back to topReviewer CommentsReviewed by: Luis Antonio Parada, CIC Biogune, Derio, Spain.In my experience the primers are better preserved when kept at
Multicolour－3DFISH－in－vertebrate－cells5
Author NotesAfter the fourth round of DOP amplification the probe quality is considerably reduced.Use the low stringency cycles only in case you start
Multicolour－3DFISH－in－vertebrate－cells3
Pepsin treatmentEquilibrate slides (kept in 50%FA/2xSSC) in 2xSSC 2 minutes;Switch to 1xPBS 3 minutes;Pepsin: 3-5 minutes in 0.01 N HCl/0.002% pepsin.
Multicolour－3DFISH－in－vertebrate－cells2
Small DNA-probes from cosmids or plasmids clonesThese kind of probes, especially plasmids, have become out of fashion for 3D-FISH due to their delicat
Multicolour－3DFISH－in－vertebrate－cells4
DetectionThe choice of the detection scheme depends on several factors: (i) the number of haptens and fluorochromes used for probe labeling; (ii) the
Differentiate－ES－cells－into－glial－cells－and－neurons
Day -1: Pass ES cells at normal density on gelatinized plate to free the culture of contamination fibroblast cells.___________________Day 1: Trypsiniz
Culture－of－BEND－Cells－(Bovine－Endometrial－Cells)
Culture of BEND Cells (Bovine Endometrial Cells)Charles E. Krininger, III and Peter J. Hansen Dept. of Animal Sciences, University of FloridaThis prot
Differentiating－Neural－Stem－Cells－into－Neurons－and－Glial－Cells
实验概要The protocols in this section describe the steps involved in differentiating neural stem cells (NSC) to neurons, astrocytes, and oligodendrocyte
Trypsinizing－cells
There are many procedures with which to trypsinize cells. All include washing the cell monolayer with TD, or in rare cases, with VE. This removes seru
Lyophilizing－Cells
Lyophilizing CellsInoculate 200 ml L-broth supplemented with appropriate antibiotics with the bacteria to be lyophilized.Incubate the culture at 37°C
Growing－cells
No two cell lines behave exactly the same, so you must learn the peculiarities, or personality, of each of the cell lines with which you work. Irrespe
Lyophilizing－Cells
Inoculate 200 ml L-broth supplemented with appropriate antibiotics with the bacteria to be lyophilized.Incubate the culture at 37°C with vigorous shak
Freezing－Cells
1) Keep prepared solutions on ice.2) Determine total cell count of cells to be frozen. (e.g. 1 X 108 )3) Determine number of vials to be frozen. (e.g.
Isolation－of－papillary－cells
Isolation of renal papillary cells1. For isolation of papillary cells, kidneys were harvested and kept in HBSS containing 15 mM HEPES, penicillin/
Fluorescent－Staining－of－Cells
1. Fluorescent phalloidin in methanol. Phallacidin does not work as well. Dilute 10 ul 330 nM stock into 500 ul PBS for each large coverslip.2. PB
Preparing－cells－and...
实验概要The method provides a protocol and tips for BrdU staining in tissue sections.Bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU) is a synthetic
Collection－of－Peritoneal－Cells
Prepare a 10ml syringe fitted with a 26G short needle and filled with 5 to 7 ml of medium and 2 to 3 ml of air. Air is important.Prepare a Pasteur pip
Immunofluorescence－Labeling－of－Cells
实验概要Antibodies are an important tool for demonstrating both the presence and the subcellular localization of an antigen. Cell staining is a very ver
Electroporation－of－ES－cells
Cells are routinely passaged two days prior to electroporating. Usually one 10 cm plate at approximately 80% confluency will provide enough cells for
Subculturing－Adherent－Cells
实验概要The following protocol describes a general procedure for subculturing adherent mammalian cells in culture.主要试剂1. Complete growth medium, pre-warme
KARYOTYPING－ES－CELLS
An actively growing culture of cells is required, i e 2 - 3 d ES cell culture. The total number of cells needs to be between 106 - 107 cells.N B Read
Transfecting－Suspension－Cells
实验概要将转移基因整合到细胞染色体DNA上,形成稳定表达转移基因的细胞系。 实验原理 细胞转染技术是目前广泛应用于病毒基因结构与功能以及基因调控等的研究。细胞转染可分为短暂转染和稳定(或永久) 转染两种。在短暂转染中,被转染基因并不整合至细胞染色体中,因而不能随细胞分裂而传代。转入病毒基因的转
Freezing－and－Thawing－cells
Freezing and Thawing cellsFreezingIt is best to freeze cells that are growing rapidly. With adherent cells, it is easiest to set up 100 mm dishes, giv
Isolation－of－papillary－cells
实验概要This protocols provides a general protocol for isolation of papillary cells.实验步骤Isolation of renal papillary cells1. For isolation of papillary c
Collection－of－Peritoneal－Cells
Prepare a 10ml syringe fitted with a 26G short needle and filled with 5 to 7 ml of medium and 2 to 3 ml of air. Air is important.Prepare a Pasteur pip
Freezing－cells－in－liquid－nitrogen
Take off MediaTrypsinate with 1ml x2 Dulbecco A trypsinAdd 7ml MediaPipette up and down to distribute cells throughout media (i.e. not clumped togethe
Belcher/Knight:－Electrocompetent－Cells
Electrocompetent CellsFrom Danijela Dukovski at Harvard Medical School. This protocol works well. --Julie NorvilleMaterialsDI water10% GlycerolSpecial
Preparing－chemically－competent－cells
MaterialsPlate of cells to be made competentTSS bufferLB mediaIceGlassware & EquipmentFalcon tubes500μl Eppendorf tubes, on ice200ml conical flask200μ
Routine－Culturing－of－ES－Cells
Cell are normally passaged every 2-3 days, this is important to avoid differentiation.Signs of differentiation are:-i) colonies are surrounded by flat