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SDDRC - Classical staining techniques and multiplex immunofluorescence

Continue your microscopy education with the San Diego Digestive Research Center's second lecture, focusing on histology samples and best practices for your research workflow. Learn to maximize the information you can extract from classical stains like H&E and PSR/FG, get guidance on designing effective multiplexing experiments, and explore the latest advances in quantitative microscopy. Perfect for researchers looking to incorporate these techniques into their work. Learning Objectives Master the Fixation Process: Understand why "garbage in equals garbage out" and how to optimize formalin timing, volume, and temperature. Navigate Tissue Processing: Learn the steps of dehydration, clearing, and paraffin infiltration to ensure samples cut smoothly and stay on the slide. Optimize Organ Preparation: Compare gut preparation techniques, including longitudinal strips and the "Swiss Roll" method, to minimize sampling errors. Solve Multiplexing Challenges: Learn to manage autofluorescence through chemical bleaching and avoid spectral bleed-through by choosing the right filter sets. Validate Antibodies Rigorously: Move beyond simple IgG controls to biological validation using knockout tissues, cell pellets, and RNA expression data. Chapters 00:00 – Introduction to Multiplex Immunofluorescence 01:08 – Visualizing Dynamic Biology in Fixed Tissues 02:22 – The Golden Rule of Histology: Fixation First 03:20 – Characteristics of an Ideal Fixative 05:39 – Formalin, PFA, and the Chemistry of Formaldehyde 07:19 – Safety First: Why You Should Never Smell the Fixative 08:08 – Trimming, Volume Ratios, and Fixation Timing (24-72 Hours) 09:33 – Best Practices for Containers: Why Flat Bottoms Matter 10:07 – Labeling Etiquette: Pencil vs. Sharpie 10:48 – SDDRC Submission Portal and Unique Identifiers 11:42 – Handling Samples and the "No Bouin's" Policy 13:09 – Sampling Errors: Understanding Continuous vs. Skip Lesions 14:37 – Gut Prep: Longitudinal Strips and Biopsy Sponges 15:39 – The Swiss Roll: Using 3D-Printed Aids for Neater Edges 16:54 – Case Study: Troubleshooting Under-Fixed Liver Samples 20:56 – Evaluating Quality: Recognizing "Mushy" vs. Crisp Morphology 23:37 – Cryopreservation: OCT, Heat Extractors, and Isopentane 25:27 – Preserving Fluorescent Proteins 26:54 – Avoiding Inclusion Bodies and Protein Overexpression Stress 28:22 – The Workflow of Tissue Processing (Dehydration to Paraffin) 30:08 – The Art of Embedding and Sectioning 31:51 – Slide Selection: Adherence, Expiration, and Cleanliness 33:01 – Archival Samples and the 150-Year History of H&E 35:21 – Transitioning to Molecular Diagnostics and Spatial Transcriptomics 36:39 – Bright-Field Analysis: White Balance and Stain Separation 38:53 – Fluorescence Fundamentals: Filter Cubes and Dichroic Mirrors 41:02 – The Triple Threat: Autofluorescence, Cross-Reactivity, and Channel Limits 42:22 – Strategies for Autofluorescence: Quenching vs. LED Bleaching 43:32 – Multiplexing Logic: Primary Species and Secondary Antibodies 44:51 – Troubleshooting Spectral Bleed-through in Multi-Band Filters 46:56 – Multiplex Platforms: SignalStar, Sequential Staining, and RareCyte Orion 48:49 – SDDRC Staining Workflow: Reusable Cover Plates and Cost Saving 49:54 – Antibody Validation: Using Knockouts and Cell Pellets 52:42 – Final Steps: Counterstaining, Mounting, and #1.5 Coverslips 53:50 – Conclusion and Upcoming SDDRC Training Courses Expert Histology Tips: Never Use Sharpie on Cassettes: Standard permanent markers will dissolve during tissue processing. Always label your cassettes with a pencil or a specialized histology "stat-mark" pen. The 20:1 Rule: Always ensure your fixative volume is at least 20 times the volume of the tissue. Fixatives are inexpensive, but your samples are not—don't try to save money by using small volumes. Ditch the Conical Tubes: Fixing organs in narrow 15ml or 50ml conical tubes causes the tissue to assume the shape of the tube and prevents the fixative from reaching the side touching the plastic. Use flat-bottom jars or fix on a rocker. Use #1.5 Coverslips Only: High-resolution microscope objectives are specifically designed for the thickness of a #1.5 coverslip. Using #1 or #2 coverslips will introduce optical aberrations that degrade your image quality. Biological Over-Technical Controls: A "no primary" control only tells you about your secondary antibody. To know if your staining is real, always include a biological negative control (such as a knockout tissue or a cell line with no RNA expression of the target). Notes and links to publications and resources: https://docs.google.com/document/d/1K...