days Protocols

Category: Reporter Gene Assays: Luciferase Assay Protocols

Protocol describes a split luciferase complementation assay that can be used to repetitively and noninvasively study the interaction of proteins in small living animals. After the expression of the appropriate vectors has been checked in cell culture in vivo, studies can be performed either by implanting transiently transfected cells for short-term analysis (maximum of 7 days), or with tumor models grown from tumor cells stably expressing the complete reporter system. - [Read Split Luciferase Complementation Assay for Studying Interaction of Proteins X and Y in Living Mice]

Category: Chromatography Protocols: Liquid Chromatography Protocols

Protocol for stable isotope labeling by amino acids in cell culture (SILAC). Protocol describes how to apply SILAC and the use of nano-scale liquid chromatography coupled to electrospray ionization mass spectrometry for protein identification and quantification.  This procedure can be completed in 8 days. - [Read Stable Isotope Labeling by Amino Acids in Cell Culture SILAC Protocol]

Category: Primary Cell Isolation and Culture Protocols

This protocol describes a method for static culture of early postimplantation mouse embryos on a microscope stage. Embryos between 6.5 and 9.5 days post coitum (dpc) can be cultured and imaged for 24 hours, with very little growth retardation. - [Read Static Culture of Postimplantation Embryos for Imaging Protocol]

Category: Mouse Protocols: Mouse Cell Culture Protocols

Protocol describes a method for static culture of early postimplantation mouse embryos on a microscope stage. Embryos between 6.5 and 9.5 days post coitum (dpc) can be cultured and imaged for 24 hours, with very little growth retardation. - [Read Static Culture of Postimplantation Embryos for Imaging Protocol]

Category: Cell Culture Protocols

Protocol describes static culture of postimplantation embryos, an alternative to the roller method. The static method is best suited to 6.0 to 7.0 days post coitum (dpc) embryos followed for 24 hours (7.0 dpc embryos) to 48 hours (6.0 dpc embryos) of development. It allows repetitive real-time observation with minimal handling of the embryo. It is especially useful if single or small groups of embryos need to be distinguished from each other. - [Read Static Culture of Postimplantation Embryos Protocol]

Category: Transfection Protocols: Calcium Phosphate Transfection Protocols

This protocol describes two transfection methods for expressing GFP-tagged actin in primary neurons. The lipid reagent DOTAP (Roche Diagnostics) method produces actin-GFP-expressing hippocampal neurons that survive well during long periods in culture. The calcium phosphate method can be used to transfect neurons that have already been growing on coverslips in vitro. Transfected cells suitable for imaging can be obtained in cultures up to 15 days in vitro. - [Read Transfecting Cultured Hippocampal Neurons with an Actin-GFP Plasmid]

Category: Epigenetics and Methylation Protocols: 5-Aza-dC Treatment AZA Procedure

"Cells were seeded at a density of 1X106 cells/100-mm dish with 10% FBS and allowed to attach over a 24-h period. 5-Aza-dC (Sigma) was then added to a final concentration of 0.2–1
M, and the cells were allowed to grow for 5 days. The medium with or w - [Read Treatment of Cells with 5-aza-dC. protocol PDF]

Category: Fungi Protocols: Neurospora Protocols

Simple method for growing Neurospora and for isolation of DNA that may be performed in two days from start to finish. The growth of mycelia in Petri plates eliminates the need for large numbers of flasks when growing many cultures for DNA isolation. - [Read Ultra-Fast Method of DNA Extraction from Neurospora Protocol]

Category: Mouse Protocols: Mouse Reproductive System Surgery Protocols

Protocol describes a procedure for uterine transfer, which is used for chimera production. The method is based on extensive work which resulted in the first successful development and birth of in-vitro-cultured mouse embryos. It is best to practice this procedure first on a cadaver and then on an anesthetized 2.5-days post coitum (dpc) pseudopregnant mouse using blue Affigel beads rather than embryos. - [Read Uterine Transfer Protocol]

Category: Mouse Protocols: Mouse Reproductive System Surgery Protocols

Protocol describes a method for vasectomy in which the vas deferens is accessed through the abdominal wall. Mice are ready for mating after ~10-14 days. Vasectomized males can be bred with fertile females to obtain plugs for timed matings. The pseudopregnant females can then be used for oviduct and uterine transfers. For an alternative protocol, see Vasectomy for Generation of Sterile Males: Access via Scrotal Sac. - [Read Vasectomy for Generation of Sterile Males: Access via Abdominal Wall Protocol]

Category: Mouse Protocols: Mouse Reproductive System Surgery Protocols

Protocol describes a method for vasectomy in which the vas deferens is accessed through the scrotal sac. Mice are ready for mating after ~10-14 days. Vasectomized males can be bred with fertile females to obtain plugs for timed matings. The pseudopregnant females can then be used for oviduct and uterine transfers. - [Read Vasectomy for Generation of Sterile Mouse Males: Access via Scrotal Sac Protocols]

Category: RNAi Technology Protocols: RNAi Avian Embryos Protocols

Protocol describes a method for staining nerve fibers in whole-mount preparations of avian embryos using an antibody against the 160-kD subunit of neurofilaments.  This allows the comparison of the branching pattern of motor and sensory neurons between control and experimental embryos.  This protocol has been successfully applied for embryos at different stages up to about stage 33 (7 days of incubation). - [Read Whole-Mount Preparations for Analysis of Axonal Pathfinding in dsRNA-Treated Avian Embryos]

Category: Avian Bird Protocols

Protocol describes a method for staining nerve fibers in whole-mount preparations of avian embryos using an antibody against the 160-kD subunit of neurofilaments. This allows the comparison of the branching pattern of motor and sensory neurons between control and experimental embryos. This protocol has been successfully applied for embryos at different stages up to about stage 33 (7 days of incubation). - [Read Whole-Mount Preparations for Analysis of Axonal Pathfinding in dsRNA-Treated Avian Embryos Protocol]