Peroxisomes: Methods and Protocols

Description

This fully updated volume assembles a comprehensive collection of methods, techniques, and strategies to investigate the molecular and cellular biology of peroxisomes in different organisms. Peroxisome research is on the rise, as novel functions and proteins of this dynamic organelle are still being discovered through studies in model systems including humans, mice, flies, plants, fungi, and yeast, and this progress is reflected in the chapters included in this collection. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.  Authoritative and up-to-date, Peroxisomes: Methods and Protocols, Second Edition serves as an ideal guide for researchers working on peroxisome- and organelle-based research questions. Dedication Preface Contents Contributors Chapter 1: Isolation of Mammalian Peroxisomes by Density Gradient Centrifugation 1 Introduction 2 Materials 2.1 General Materials 2.2 Isolation of Rat Liver Peroxisomes 2.3 Separation of Peroxisomes from HepG2 Cells 3 Methods 3.1 Isolation of Peroxisomes from Rat Liver 3.2 Purification of Peroxisomes from HepG2 Cells 4 Notes References Chapter 2: Analysis of Yeast Peroxisomes via Spatial Proteomics 1 Introduction 2 Materials 2.1 Yeast and Culture Media 2.2 Cell Lysis and Preparation of Spheroplasts 2.3 Subcellular Fractionation 2.3.1 Differential Centrifugation 2.3.2 Nycodenz Density Gradient Centrifugation 2.4 LC-MS Sample Preparation 2.4.1 Acetone Precipitation 2.4.2 Proteolytic In-Solution Digestion 2.4.3 Desalting of Peptides 2.5 LC-MS/MS Analysis 2.6 Data Analysis 3 Methods 3.1 Yeast Cell Culture and Harvest 3.2 Cell Lysis and Generation of Spheroplasts 3.3 Subcellular Fractionation 3.3.1 Preparation of Cellular Fractions by Differential Centrifugation 3.3.2 Generation of a Gradient-Purified Peroxisomal Fraction by Nycodenz Density Gradient Centrifugation 3.4 LC-MS Sample Preparation 3.4.1 Acetone Precipitation 3.4.2 Proteolytic In-Solution Digestion 3.4.3 Desalting of Peptides and Preparation of Samples for LC-MS Analysis 3.5 LC-MS Analysis 3.6 Data Analysis 4 Notes References Chapter 3: Isolation of Glycosomes from Trypanosoma brucei 1 Introduction 2 Materials 2.1 Cell Culture 2.2 Reagents and Solutions 2.3 Materials 3 Methods 3.1 Cultivation of T. brucei Cells and Harvesting 3.2 Rupture of the Cells with Silicon Carbide 3.3 Preparation of an Organelle Enriched Fraction by Differential Centrifugation 3.4 Isolation of Glycosomes by Density Gradient Centrifugation 3.5 Successive Enrichment of Glycosomal Membrane Proteins 4 Notes References Chapter 4: Immunolabeling for Detection of Endogenous and Overexpressed Peroxisomal Proteins in Mammalian Cells 1 Introduction 2 Materials 2.1 Mammalian Cells and Plasmids 2.2 Cell Culture Equipment 2.3 Cell Culture Media, Buffers, and Reagents 2.4 Transfection 2.5 Immunofluorescence and Fluorescence Microscopy 2.6 Controls 2.7 Antibody Sources 3 Methods 3.1 Cell Culture 3.2 Transfection of Human Skin Fibroblasts (dMFF) Using Microporation (Optional) 3.3 Detection of Expressed (or Endogenous) Peroxisomal Proteins 4 Notes References Chapter 5: Super-Resolution Imaging of Peroxisomal Proteins Using STED Nanoscopy 1 Introduction 2 Materials 2.1 Mammalian Cells and Culturing 2.1.1 Media and Buffers 2.1.2 Equipment 2.2 Yeast Cells and Cultivation 2.2.1 Media and Buffers 2.2.2 Equipment 2.3 (Live-Cell) Dyes and Antibodies (See Notes 5 and 6) 2.4 Fixed Sample Preparation for Confocal or STED Nanoscopy 2.5 STED Nanoscopy 3 Methods 3.1 Seeding HEK Cells for Immunofluorescence 3.2 Immunofluorescence of Fixed HEK Cells for STED Nanoscopy 3.3 HaloTag or SNAP-Tag Labeling of Mammalian Cells for STED Nanoscopy 3.3.1 Live-Cell Labeling of HEK Cells 3.3.2 Fixed-Cell Labeling of HEK Cells with Live Dyes 3.4 HaloTag or SNAP-Tag Labeling of Fixed Yeast Cells for STED Nanoscopy 3.5 STED Nanoscopy 4 Notes References Chapter 6: Direct Stochastic Optical Reconstruction Microscopy (dSTORM) of Peroxisomes 1 Introduction 2 Materials 2.1 Immunofluorescence. 2.2 Super-Resolution Imaging 3 Methods 3.1 Immunofluorescence 3.2 Super-Resolution Imaging 4 Notes References Chapter 7: Correlative Light- and Electron Microscopy in Peroxisome Research 1 Introduction 2 Materials 2.1 Buffers and Solutions 2.2 Equipment 2.3 Software 3 Methods 3.1 Fixing the Yeast Cells 3.2 Gelatin Embedding and Cryo-Protection 3.3 Preparation of Gold Decorated Grids 3.4 Cryo-Sectioning 3.5 Fluorescence Microscopy 3.6 Transmission Electron Microscopy and Image Alignment 4 Notes References Chapter 8: Ultrastructural Analysis and Quantification of Peroxisome-Organelle Contacts 1 Introduction 2 Materials 2.1 Cell Culture 2.1.1 Mammalian Cells 2.1.2 Cell Culture Equipment 2.1.3 Cell Culture Media and Reagents 2.2 Processing and Embedding of Cultured Cells for TEM 2.3 Ultrathin Sectioning and Contrasting of TEM Sections 2.4 TEM Imaging and Stereological Analysis 3 Methods 3.1 Cell Culture 3.2 Primary Fixation of Cell Monolayers 3.3 Sample Processing and Embedding 3.4 Ultramicrotomy and Contrasting of TEM Sections 3.5 TEM Analysis and Sampling Procedures 3.6 Spatial Stereology 4 Notes References Chapter 9: Detection of Peroxisomal Proteins During Mycobacterial Infection 1 Introduction 2 Materials 2.1 Culturing Mycobacterium sp. 2.1.1 Solid and Liquid Culturing Media 2.1.2 Consumables 2.1.3 Instruments 2.2 Cell Culture 2.2.1 Immunofluorescence Microscopy 3 Methods 3.1 Culturing of Mycobacterium sp. 3.1.1 Preparation of Middlebrook 7H9 Liquid Culture Media 3.1.2 Culturing M. tb in Middlebrook 7H9 Liquid Culture Media Safety Precautions Culture Methods 3.1.3 Preparation of Solid Middlebrook 7H11 Agar Plates 3.1.4 Culturing M. tb on Solid Mycobacteria 7H11 Agar Plates 3.1.5 Preparation of M. tb Glycerol Stocks 3.2 Immunofluorescence Microscopy 3.2.1 Isolation of Bone Marrow-Derived Macrophages (BMDMs) 3.2.2 Seeding of Macrophages Calculation of Seeding of BMDMs 3.2.3 Bacterial Infection 3.2.4 Microscopic Slide Preparation 4 Notes References Chapter 10: Proximity-Ligation Assay to Detect Peroxisome-Organelle Interaction 1 Introduction 2 Materials 2.1 Mammalian Cells and Plasmids 2.2 Cell Culture Equipment 2.3 Cell Culture Media, Buffers, and Reagents 2.4 Transfection 2.5 Proximity Ligation Assay (PLA) 2.6 Controls 2.7 Primary Antibodies 3 Methods 3.1 Cell Culture 3.2 DEAE-Dextran Transfection of COS-7 cells (see Notes 14 and 15) 3.3 Detection of Protein Proximity by PLA 3.4 Quantification of Peroxisome-Organelle/ER Contacts 4 Notes References Chapter 11: Assay of Reactive Oxygen/Nitrogen Species (ROS/RNS) in Arabidopsis Peroxisomes Through Fluorescent Protein Contain... 1 Introduction 2 Materials 2.1 Plant Samples 2.2 Fluorescent Probes 2.3 Confocal Laser Scanning Microscopy (CLSM) 3 Methods 4 Notes References Chapter 12: Identification of Peroxisome-Derived Hydrogen Peroxide-Sensitive Target Proteins Using a YAP1C-Based Genetic Probe 1 Introduction 2 Materials 2.1 Proteomics Sample Preparation 2.1.1 Equipment 2.1.2 Compounds and Materials for Keratin Removal from Surfaces and Objects 2.1.3 Other Materials 2.1.4 Compounds and Buffers 2.2 Cell Culture and Cell Treatment 2.3 Electrophoretic Mobility Shift Assay and Immunoblotting 2.4 LC-MS/MS 3 Methods 3.1 Measures to Reduce Keratin Contamination 3.2 Cell Culture 3.2.1 Maintenance of the Cells 3.2.2 Pretreatment of po-DD-DAO Flp-In T-REx 293 Cells 3.3 Sample Preparation 3.3.1 Preparation of Cell Suspension 3.3.2 Cell Treatment 3.3.3 Preparation of a Cleared Cell Lysate 3.3.4 Enrichment of IBD-SBP-YAP1C and IBD-SBP-YAP1C-Trapped Proteins on a Streptavidin Affinity Matrix 3.3.5 Removal of the Non-bound Proteins from the Streptavidin Affinity Matrix 3.3.6 Elution of the IBD-SBP-YAP1C-Trapped Proteins 3.3.7 Reduction and Alkylation of Available Thiol Groups 3.3.8 Protein Precipitation 3.3.9 Trypsin Digestion 3.3.10 Peptide Desalting 3.4 Quality Control of the Samples 3.5 Identification of IBD-SBP-YAP1C-Trapped Proteins Using LC-MS/MS 3.6 Data Analysis 4 Notes References Chapter 13: Assessment of the Peroxisomal Redox State in Living Cells Using NADPH- and NAD+/NADH-Specific Fluorescent Protein ... 1 Introduction 2 Materials 2.1 Cell Culture 2.1.1 Equipment 2.1.2 Materials 2.2 Electroporation 2.3 Fluorescence Microscopy 3 Methods 3.1 Cell Culture 3.2 Neon Electroporation of Flp-In T-REx 293 Cells 3.2.1 Preparation of the Cells 3.2.2 Electroporation of the Cells 3.3 Pyridine Nucleotide Measurements and Analysis 3.3.1 Preparing for Live-Cell Imaging 3.3.2 Image Collection 3.3.3 Image Analysis 3.4 Functional Validation of po-SoNar 3.5 Functional Validation of po-iNAP1 4 Notes References Chapter 14: Live-Cell Imaging of Peroxisomal Calcium Levels and Dynamics 1 Introduction 2 Materials 2.1 Materials for Ca2+ Measurement 2.2 Setup for Live-Cell Imaging 3 Methods 3.1 Cell Culture and Cell Preparation 3.2 Measurement of Ca2+ in HeLa or Any Other Histamine-Responsive Cells 3.3 Measurement of Ca2+ in Histamine-Insensitive (or Histamine-Sensitive) Cells 3.4 Data Analysis for FRET Ca2+ Sensors 3.5 Calibration Experiments for Absolute Ca2+ Concentration 3.6 Determination of the Dissociation Constant Kd 3.7 Calculation of Absolute Ca2+ Concentration 4 Notes References Chapter 15: Analysis of Peroxisome Biogenesis by Phos-Tag SDS-PAGE 1 Introduction 2 Materials 2.1 SDS Polyacrylamide Gel Containing Phos-Tag 2.2 Transfer to PVDF Membrane 2.3 Sample Preparation 2.4 Cell Culture and Immunoblotting 2.5 Equipment 3 Methods 3.1 Casting of Phos-Tag PAGE Gel 3.2 Sample Preparation (from Cultured Cells) 3.3 Electrophoresis 3.4 Transfer to PVDF Membrane 4 Notes References Chapter 16: Targeted Modifications of the Yeast Genome to Study Peroxisomes 1 Introduction 2 Materials 2.1 Growth and Maintenance of Yeast Cells 2.2 High-Efficiency Transformation into Yeast Cells 2.3 Yeast Genomic DNA Isolation 2.4 PCR Reactions 2.5 Agarose Gel Electrophoresis 3 Methods 3.1 Oligonucleotide Design and Polymerase Chain Reaction (PCR) Amplification of Gene Replacement Cassette 3.2 High-Efficiency Transformation into Yeast Cells 3.3 Yeast Genomic DNA Isolation 3.4 Analytical PCR to Validate the Gene Deletion in the Genome 3.5 N- and C-Terminal Epitope Tagging in the Genome 3.6 Generation of Strains with Conditional Gene/Protein Expression 3.6.1 In Genome Modification for Conditional Gene Expression Using the Regulatable GAL1 Promoter 3.6.2 In Genome Modification for Conditional Gene Expression Using AID Tag for Controlled Protein Degradation 4 Notes References Chapter 17: Applying CRISPR-Cas9 Genome Editing to Study Genes Involved in Peroxisome Biogenesis or Peroxisomal Functions 1 Introduction 2 Materials 2.1 Plasmids (Available with MTA via www.addgene.org) 2.2 Reagents 2.3 PCR and Sequence Primers 2.4 Transformation of E. coli 2.5 Mammalian Cells 2.6 Equipment and Antibodies 3 Methods 3.1 Design of Guide RNAs 3.1.1 General 3.1.2 Introduction of Small DNA Sequences, Including Single-Nucleotide Variants 3.2 Cloning of gRNA-Encoding Sequences in pSPCas9 Plasmids 3.3 Transfection of pCRISPR-Cas9 Plasmids into Human Cells 3.4 Screening of Clonal Cells 3.5 Validation and Functional Studies 4 Notes References Chapter 18: Generation of Reporter Cell Lines for Endogenous Expression Analysis of Peroxisomal Proteins 1 Introduction 2 Materials 2.1 Molecular Biology 2.1.1 Reagents for Construction of the CRISPR-Cas9 Vector 2.1.2 Reagents for Construction of the CRIS-PITChv2 Donor Vector 2.1.3 Molecular Biology Equipment 2.2 Mammalian Cell Culture 2.2.1 Cell Culture Media, Buffers, and Reagents 2.2.2 Cell Culture Equipment 2.3 Transfection 2.4 Selection and Single-Cell Cloning 2.5 Genotyping Analysis and Reporter Cell Validation 3 Methods 3.1 Oligonucleotide Design 3.2 Construction of CRISPR-Cas9 Vector 3.2.1 Insertion of the Locus-Specific gRNA Oligonucleotides into the pX330A-1x2 Vector 3.2.2 Introduction of the PITCh gRNA into the pX330A-1x2 Vector Containing the Locus-Specific gRNA 3.3 Construction of the PITCh Donor Vector 3.4 Transfection and Selection to Generate Knock-in Cells 3.5 Single-Cell Cloning 3.6 Genotyping Analysis and Reporter Cell Validation 4 Notes References Chapter 19: Peroxisomes and Viruses: Overview on Current Knowledge and Experimental Approaches 1 Introduction 2 Interplay Between Peroxisomes and Viruses 2.1 Influenza A Virus (IAV) 2.2 Members of the Coronaviridae Family 2.2.1 Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) 2.2.2 Porcine Deltacoronavirus (PDCoV) and Porcine Epidemic Diarrhea Virus (PEDV) 2.3 Rotavirus 2.4 Human Immunodeficiency Virus (HIV) 2.5 Enterovirus 71 2.6 Members of the Herpesviridae Family 2.6.1 Human Cytomegalovirus (HCMV) 2.6.2 Herpes Simplex Virus 1 (HSV-1) 2.6.3 Kaposi´s Sarcoma-Associated Herpesvirus (KSHV) 2.7 Members from the Flaviviridae Family 2.7.1 Dengue Virus (DENV) 2.7.2 West Nile Virus (WNV) 2.7.3 Zika Virus (ZIKV) 2.7.4 Hepatitis C Virus (HCV) 3 Conclusions References Chapter 20: Tools to Investigate the Peroxisome-Dependent Antiviral Response 1 Introduction 2 Materials 2.1 Stimulation of the Peroxisomal MAVS Signaling 2.1.1 Transfection of Cultured Mammalian Cells with 3p-hpRNA 2.1.2 Infection of Cultured Mammalian Cells with Influenza A Virus 2.2 Analysis of the Antiviral Signaling Activation 2.2.1 SDS-PAGE and Immunoblotting 2.2.2 RNA Isolation and RT-qPCR 3 Methods 3.1 Stimulation of the Peroxisomal MAVS Signaling 3.1.1 Transfection of Cultured Mammalian Cells with 3p-hpRNA 3.1.2 Infection of Cultured Mammalian Cells with Influenza A Virus 3.2 Analysis of the Antiviral Signaling Activation 3.2.1 SDS-PAGE and Immunoblotting 3.2.2 RNA Isolation and RT-qPCR 4 Notes References Chapter 21: Determining the Importance of Peroxisomal Proteins for Viral Infections in Cultured Mammalian Cells 1 Introduction 2 Materials 2.1 Infection of Cultured Mammalian Cells with Influenza A Virus 2.2 Immunofluorescence Analysis 2.3 Plaque Assay 3 Methods 3.1 Infection of Cultured Mammalian Cells with Influenza A Virus 3.2 Immunofluorescence Analysis 3.3 Plaque Assay 4 Notes References Chapter 22: Characterization of Yeast Peroxisomes: Enrichment of Peroxisomal Fractions and Analysis of β-Oxidation Activity 1 Introduction 2 Materials 2.1 Yeast Strain 2.2 Media 2.3 Stock Solutions and Chemicals for Spheroplasting and Subcellular Fractionation 2.4 Equipment 3 Methods 3.1 Induction of Peroxisome Proliferation 3.2 Preparation of Spheroplasts 3.3 Differential Centrifugation of Spheroplast Homogenate 3.4 Nycodenz Density Gradient Centrifugation 3.4.1 Preparation of Solutions 3.4.2 Preparation of a Nycodenz Gradient 3.4.3 Gradient Centrifugation 3.5 Markers for Intracellular Compartments 3.5.1 Peroxisomes: 3-Hydroxyacyl-CoA Dehydrogenase (3-HAD) 3.5.2 Mitochondria: Fumarase 3.5.3 Cytosol: Phosphoglucose Isomerase (PGI) 3.6 Preparation of Yeast Lysates 3.7 Peroxisomal β-Oxidation Activity in Intact Yeast Cells 3.8 Special β-Oxidation Assays in Isolated Peroxisomes or Lysates 3.8.1 [1-14C] Acyl-CoA β-Oxidation Activity in Isolated Peroxisomes 3.8.2 [1-14C] Acyl-CoA β-Oxidation Activity in Lysates 4 Notes References Chapter 23: A Cell-Free In Vitro Import System for Peroxisomal Proteins Containing a Type 2 Targeting Signal (PTS2) 1 Introduction 2 Materials 2.1 Preparation of Rat Liver Post-nuclear Supernatant (PNS) 2.2 In Vitro Synthesis of Proteins 2.3 In Vitro Import Experiments 3 Methods 3.1 Preparation of the Post-nuclear Supernatant (PNS) 3.2 In Vitro Synthesis of Proteins 3.3 In Vitro Import Experiments 4 Notes References Chapter 24: Assessing Peroxisomal Protein Interaction by Immunoprecipitation 1 Introduction 2 Materials 2.1 Cell Culture and Plasmids 2.2 Immunoprecipitation (IP) 2.3 SDS-PAGE and Western Blotting 2.4 Antibody Sources and Detection 3 Methods 3.1 Cell Culture and Transfection 3.2 Preparation of Cell Lysates 3.2.1 Cell Scraping 3.2.2 Cell Trypsinization 3.2.3 Clearing of Lysates 3.3 Preparation of Beads 3.4 Immunoprecipitation 3.5 Detecting Protein Interactions by Western Blotting 4 Notes References Chapter 25: Purification of a Recombinant Human PEX1/PEX6 AAA+ ATPase Complex from HEK293TT Cells 1 Introduction 2 Materials 2.1 Cell Culture 2.2 PEI Lipofection 2.3 Buffers and Chemicals 2.4 Cell Lysis 2.5 Protein Purification Essentials 2.6 SDS-PAGE 2.7 Silver Staining 2.8 Western Blot and Immunodetection 3 Methods 3.1 PEI Lipofection: Transfection of HEK293TT Cells 3.2 Cell Harvest and Flow Cytometry 3.3 Cell Lysis 3.4 Protein Purification 3.5 SDS PAGE 3.6 Silver Staining 3.7 Western Blot and Immunodetection 4 Notes References Chapter 26: Affinity Purification of Soluble and Membrane-Bound Protein Complexes by a FlpIn Strategy 1 Introduction 2 Materials 2.1 Cell Line 2.2 Cultivation Media and Solutions 2.3 Buffers for Soluble Complex Isolation 2.4 Buffers for Non-soluble Membrane Complex Isolation 2.5 Protease Inhibitors 2.6 Equipment and Other Materials 3 Methods 3.1 Cell Culture Conditions 3.2 Large-Scale Cultivation of Cells 3.3 Harvesting Cells from Triple Desk Flasks 3.4 Cell Lysis 3.5 Ultracentrifugation 3.6 Determination of Protein Concentration 3.7 Solubilization of Membrane-Bound Complexes 3.8 Ultracentrifugation 3.9 Affinity Chromatography 3.10 TEV Cleavage and Elution 4 Notes References Chapter 27: Utilization of Nonstop mRNA to Assess Ribosome-Associated Nascent Polypeptide Chains in Early Topogenesis of Perox... 1 Introduction 2 Materials 2.1 Yeast Strain and Vectors 2.2 Cultivation Media 2.3 Buffers for Soluble Complex Isolations 2.4 Buffers for Membrane Complex Isolation 2.5 Protease Inhibitors 2.6 Equipment and Other Materials 3 Methods 3.1 Cultivation of Yeast Cells 3.2 Sedimentation 3.3 Cell Disruption 3.4 Ultracentrifugation 3.5 Preparation of Soluble Samples 3.6 Preparation of Membrane Samples 3.7 Isolation of Soluble and Membrane Complexes 4 Notes References Chapter 28: Computational Evaluation of Peroxisomal Targeting Signals in Metazoa 1 Introduction 2 PTS1 Analysis 3 Subsequent Analyses 4 Example 5 Notes References Chapter 29: Computational Approaches for Peroxisomal Protein Localization 1 Introduction 2 Materials 2.1 Use Case-Specific Tools (See Note 1) 2.2 General Tools for Subcellular Localization and Transmembrane Detection (See Note 1) 3 Methods 3.1 Detection of Peroxisomal Protein Candidates 4 Notes References Chapter 30: Estimating the Interaction Strength Between PTS1-Peptides and Their Receptor PEX5 in Living Cells Using Flow-Cytom... 1 Introduction 2 Materials 2.1 Cells and Media 2.2 Transfection Reagent 2.3 FRET Pair (Combination of Fluorescent Proteins Capable of FRET) 2.4 Plasmids 2.5 Measurement Equipment 3 Methods 3.1 Cell Preparation and Transfection 3.2 Pre-experiment 1: FRET Efficiency of Fusion Protein 3.3 Pre-experiment 2 - Optimize Transfection Ratio 3.4 flowFRET Experiments 3.5 Pretreatment and Extraction of Data 3.6 Calculation of Donor- and Acceptor Levels and DFRET Values 3.7 Fitting and Interpretation of Results 4 Notes References Chapter 31: Using the Superfolder GFP (sfGFP) System to Study Plant Peroxisomal Protein Import 1 Introduction 2 Materials 2.1 Solutions for Protoplast Preparation 2.2 Seed Sterilization 2.3 Murashige and Skoog Medium (MS) 2.4 sfGFP11 Plasmids Preparation 2.5 Transgenic Lines that Express sfGFP1-10OPT 2.6 Equipment 3 Methods 3.1 Seed Sterilization 3.2 Seed Sowing 3.3 Seed Transplanting 3.4 Protoplast Isolation 3.5 Protoplast Transfection Assay 3.6 Microscopy 4 Notes References Chapter 32: Biochemical Fractionation of Trypanosomes for the Analysis of Glycosomal Protein Import Defects 1 Introduction 2 Materials 2.1 Trypanosoma brucei (Bloodstream form, BSF) Culture 2.2 Reagents and Buffer Solutions for Cell Collection, Protein Estimation and Fractionation 2.3 Standard Equipment 3 Methods 3.1 Trypanosoma brucei Culture and Cell Harvesting 3.2 Protein Estimation 3.3 Digitonin Fractionation 3.4 Other Application of the Digitonin Fractionation Protocol 4 Notes References Chapter 33: Manipulation and Visualization of Peroxisomes in Drosophila 1 Introduction 2 Materials 2.1 S2 Cell Culture 2.2 Transfection Reagents and Multiwell Dishes 2.3 S2 Cell/Tissue Fixation for Indirect Immunofluorescence Labelling 2.4 Generation of dsRNAs Targeting Peroxisome Genes in S2 Cells 2.5 Dissecting Tissues from Drosophila Larvae, Pupa, or Adults 3 Methods 3.1 S2 Cell Culture 3.2 dsRNA Synthesis 3.3 Treating S2 Cells with dsRNAs and Monitoring RNAi Efficiency 3.4 Preparation and Transfection of S2 Cells for Live Imaging 3.5 Coating Coverglass with Concanavalin A 3.6 Transfection for Fixed Cells and Immunofluorescence Labeling 3.7 Preparation of S2 Cells Adhered to Coverglasses for Fixation and Immunofluorescence Labeling 3.8 Dissecting Tissues from Drosophila Larvae, Pupa, or Adults for Immunofluorescence Protein Detection 4 Notes References Chapter 34: Mouse Models to Study Peroxisomal Functions and Disorders: Overview, Caveats, and Recommendations 1 Introduction 2 Overview of Mouse Models 2.1 Peroxisome Biogenesis: Global Knockout Mice with Combined PTS1 and PTS2 Import Defect 2.2 Peroxisome Biogenesis: Conditional Knockout Mice with Combined PTS1 and PTS2 Import Defect 2.3 Peroxisome Biogenesis: Selective PTS2 Import Defect (Pex7 Models) 2.4 Peroxisome Biogenesis: Peroxins and Other Proteins Involved in Peroxisomal Proliferation/Fission 2.5 Peroxisomal α-Oxidation Defects 2.6 Peroxisomal β-Oxidation Defects: Global Knockouts 2.6.1 Acox1 and Acox2 2.6.2 The Multifunctional Proteins 2.6.3 The Thiolytic Cleavage Enzymes 2.6.4 Auxiliary Proteins of β-Oxidation 2.6.5 Validation of β-Oxidation Inactivation 2.7 Peroxisomal β-Oxidation Defects: Conditional Knockouts 2.8 Ether Lipid Synthesis Defects 2.9 Membrane Proteins Involved in Transport 2.10 Other Peroxisomal Matrix Proteins 3 Conclusions and Perspectives References Index