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]
With this protocol, transcripts that were initiated from specific genes by RNA polymerases prior to permeabilization can be measured. Instead of a nuclear extract, permeabilized cells are used. Includes information on: Permeabilization of Cells; In vitro Transcription Reaction (Run-off); Isolation of RNA; Preparation of Slot Blot Membrane for Hybridization; Hybridization of Nitrocellulose Membrane; TCA Precipitation to Determine Incorporation of [32P] GTP into Nucleic Acid - [Read In Vitro Transcription Assay (Run-off Assay) using Permeabilized Cells]
The water soluble, DNA intercalator, propidium iodide (PI), is used to bind to DNA after permeabilization of cells with NP40. The amount of dye bound correlates with the content of DNA within a given cell. Once cells are stained, they are analyzed on a flow cytometer. The relative content of DNA indicates the distribution of a population of cells throughout the cell cycle. - [Read Quantification of Apoptosis and Cell Cycle Distribution of Primary B Cells Using Propidium Iodide]
The water soluble, DNA intercalator, propidium iodide (PI), is used to bind to DNA after permeabilization of cells with NP40. The amount of dye bound correlates with the content of DNA within a given cell. Once cells are stained, they are analyzed on a flow cytometer. The relative content of DNA indicates the distribution of a population of cells throughout the cell cycle. - [Read Quantification of Apoptosis and the Cell Cycle Distribution of Primary B Cells Using PI]
Protocol for whole-mount in situ hybridization for the detection of RNA in C. elegans embryos. Includes: Collection of Embryos; Permeabilization and fixation of embryos; Prehybridization; Hybridization; Post-hybridization washes; Probe Detection; Double-labeling; Interpretation. - [Read Whole-Mount In Situ Hybridization for the Detection of RNA in C. elegans Embryos Protocol]