Multicolour3DFISHinvertebratecells3
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. (prepare 49.5ml H2O + 0.5ml 1NHCl at 37°C and add 10ml pepsin solution (stock solution 10%) just before use);2x5 minutes PBS/MgCl2 (95ml 1xPBS/5ml 1M MgCl2) for inactivation of pepsin;Post-fixation in 1% PFA/1xPBS 1 minute;Wash 1x5 minutes PBS;Wash 2x5 minutes in ......閱讀全文
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
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－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－cells1
IntroductionMulticolour 3D-FISH in combination with confocal microscopy, 3D image reconstruction and quantitative image analysis is an efficient tool
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－cells6
Back to topReviewer CommentsReviewed by: Luis Antonio Parada, CIC Biogune, Derio, Spain.In my experience the primers are better preserved when kept at
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
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
Differentiating－Neural－Stem－Cells－into－Neurons－and－Glial－Cells
實驗概要The protocols in ?this section describe the steps involved in differentiating neural stem ?cells (NSC) to neurons, astrocytes, and oligodendrocyte
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.
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
Inoculate 200 ml L-broth supplemented with appropriate antibiotics with the bacteria to be lyophilized.Incubate the culture at 37°C with vigorous shak
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
Lyophilizing CellsInoculate 200 ml L-broth supplemented with appropriate antibiotics with the bacteria to be lyophilized.Incubate the culture at 37°C
Isolation－of－papillary－cells
實驗概要This protocols provides a general protocol for isolation of papillary cells.實驗步驟Isolation of renal papillary cells1.?For ?isolation of papillary c
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
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
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
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/
Immunofluorescence－Labeling－of－Cells
實驗概要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
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
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
Subculturing－Adherent－Cells
實驗概要The following protocol describes a general procedure for subculturing adherent mammalian cells in culture.主要試劑1. Complete growth medium, pre-warme
Transfecting－Suspension－Cells
實驗概要將轉移基因整合到細胞染色體DNA上,形成穩定表達轉移基因的細胞系。?實驗原理? ? 細胞轉染技術是目前廣泛應用于病毒基因結構與功能以及基因調控等的研究。細胞轉染可分為短暫轉染和穩定(或永久) 轉染兩種。在短暫轉染中,被轉染基因并不整合至細胞染色體中,因而不能隨細胞分裂而傳代。轉入病毒基因的轉
Preparing－cells－and...
實驗概要The method provides a protocol and tips for BrdU staining in tissue sections.Bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU) is a synthetic
哺乳動物RNAi技術全面實驗方法
WHENVIRUSESINFECTEUKARYOTICCELLSdomly integrate into host genomes, double-stranded RNA (dsRNA) is frequently produced from the invading genes, either
Protocol－for－intracytoplasmic－staining－of－cytokines－for－FACS－analysis
DescriptionProtocol for intracytoplasmic staining of cytokines for FACS analysis?Procedure1) Prepare spleen, lymph node or T cell clone cells as singl
Isolation－of－rodent－pancreatic－β－cells
1.?Adult rats weighing 250-350g were anesthetized, sacrificed and immediately used for pancreas sampling.2.?Rat islets were isolated from male wistar
Culturing－HEK－293－Cells
ReagentsMedium:500 ml Dulbecco’s Modified Eagle Medium (Gibco #41966-029)55 ml FCS (10 %)2.8 ml Gentamycin Solution (Sigma G-1272, 10 ml))TrypsinTryps