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.

Updated sample submission guidelines: https://drive.google.com/file/d/1SQXmwyauXs-8rHwQhLRmWzr35HVKgarZ/view?usp=sharing

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 – Infinity 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 – 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 QuPath 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/1KLZ8EuaFdyON14FNWoVfFutViYp4VS_x81TWSCG5QKc/edit?usp=sharing

Видео SDDRC - Classical staining techniques and multiplex immunofluorescence канала Zbigniew Mikulski