Fundamental Approaches to Screen Abnormalities in Drosophila

ISBN-13: 9781493997558 / Angielski / Twarda / 2020 / 265 str.

Monalisa Mishra

Opis

This volume includes in-depth, hands-on protocols for detecting various developmental defects in Drosophila. It provides cutting-edge methods for maintaining Drosophila under laboratory conditions to perform various experiments, and for dissecting various imaginal discs of the larvae. Further, biochemical protocols for estimating the levels of different metabolites, reactive oxygen species, and fat-sensitive pathways are discussed, and various staining and behavior techniques for fat detection are provided.The book explains how various fluorescent dyes and the comet assay can be used to identify DNA damage, and elaborates on the analysis of the eye, antennae, imaginal disc, gut and muscle damage under bright field and fluorescent microscopes. It covers the analysis of hemolymph using Giemsa staining; determining the functionality of channel proteins, eye and mechanosensory organs in adults and larvae using various behavioral assays; and metal detection and structural analysis for various tissues using SEM. In closing, it addresses the analysis of Drosophila's eye and head via paraffin section; measurement of reactive oxygen species from various tissues via FACS; and the CRISPER technique for gene editing and analysis of micro RNA mutations.

Spis treści

Chapter 1. Preparation of normal food and modified food for Drosophila.- Chapter 2. Protocol for dissection of various tissues (gut, imaginal disc, fat body, ovary, testes) in larva and adult fly.- Chapter 3. Biochemical estimation of various metabolites (protein, lipid, carbohydrate, trehalose, cholesterol).- Chapter 4. Detection of fat within the Drosophila body.- Chapter 4.1. Nile red.- Chapter 4.2. Oil red.- Chapter 4.3. Sudan black.- Chapter 5. Estimation of reactive oxygen species (ROS) production in Drosophila cells. -Chapter 5.1. DCF-DA staining.- Chapter 5.2. NBT assay.- Chapter 6. Methods to check the metabolic activity of fly.- Chapter 6.1. Floating assay.- Chapter 6.2. Survivability assay.- Chapter 7. Detection of DNA damage in larva and adult stage of Drosophila by Fluorescent microscopy.- Chapter 8. Imaging of larva/adult gut damage using Bright field microscopy.- Chapter 8.1. Trypan blue stain.- Chapter 8.2. Prussian blue stain.- Chapter 8.3. Copper cell staining.- Chapter 8.4. Iron cell staining.- Chapter 9.Comet assay to detect severity of DNA damage.- Chapter 10. Checking the abnormality in hemolymph by Giemsa stain.- Chapter 10. Analysis of channel proteins.- Chapter 11. Analysis of channel proteins.- Chapter 11.1. Temperature sensitivity assay.- Chapter 11.2. Sound-avoidance assay.- Chapter 11.3. Hygroscopicassay.- Chapter 12.Various behavioral assays in larva and adult Drosophila.- Chapter 12.1. Larva crawling.- Chapter 12.2. Adult climbing.- Chapter 12.3. Self-righting.- Chapter 12.4.Touch response test.- Chapter 12.5. Temperature sensitivity assay.- Chapter 12.6. Thermal avoidance behavior.- Chapter 12.7. Photosensitivity assay (Light- Dark).- Chapter 12.8. Light preference test.- Chapter 12.9. Chemotaxis assay.- Chapter 12.10. Courtship and mating.- Chapter 12.11. Grooming behavior. - Chapter 12.12. Aggression assay.- Chapter 12.13. Choice assay.- Chapter 13. Detection of various metals within the tissue by Energy dispersive X-ray (EDX) through SEM and FTIR.- Chapter 14. Imaging of various sections of fly through Scanning Electron Microscopy (SEM).- Chapter 15. Preparation of thin sections and fixation of various tissues of Drosophila for examination by transmission electron microscopy (TEM).

O autorze

Dr. Monalisa Mishra is an Assistant Professor at the Department of Life Sciences, National Institute of Technology (NIT) in Rourkela, India. She completed her Ph.D. at the University of Bremen, Germany, prior to postdoctoral studies at the School of Medicine, University of San Diego, California, USA; Department of Biological Sciences, Indiana University, Bloomington, USA; and Max Plank Institute of Cell Biology and Genetics, Dresden, Germany.

Further, she previously served as an Assistant Professor at the Department of Biological Sciences, BITS in Pilani, India from 2012-2014. Currently, her lab at the NIT Rourkela is investigating various factors that can affect the development of Drosophila’s sensory organs. The lab’s findings have been published in many prominent international journals.