How can you improve sample preparation for imaging studies?

1 Choose the right fixative

The fixative is the first step in preserving the structure and function of the tissue or cells. It prevents autolysis, degradation, and contamination, and stabilizes the antigens and enzymes for immunohistochemistry or immunofluorescence. The choice of fixative depends on the type of specimen, the imaging modality, and the desired outcome. For example, formalin is widely used for light microscopy, but it can cause cross-linking and masking of some epitopes. Alcohol-based fixatives are suitable for electron microscopy, but they can cause shrinkage and distortion. Other factors to consider are the fixation time, temperature, and volume, which should be optimized for each sample.

2 Optimize the embedding and sectioning

The embedding and sectioning steps are crucial for obtaining thin and uniform slices of the specimen that can be mounted on slides or grids. The embedding medium should support the tissue or cells and provide adequate rigidity and transparency. Paraffin is the most common medium for light microscopy, but it requires deparaffinization and rehydration before staining. Resin is preferred for electron microscopy, but it requires polymerization and hardening. The sectioning method should produce smooth and intact sections that are appropriate for the imaging technique. Microtomes, cryostats, ultramicrotomes, and laser microdissection are some of the options available.

3 Select the appropriate staining

The staining step is essential for enhancing the contrast and specificity of the specimen. It allows the visualization of different structures, molecules, and interactions within the tissue or cells. The staining method should be compatible with the fixative, the embedding medium, and the imaging modality. For example, hematoxylin and eosin are standard stains for light microscopy, but they can interfere with fluorescence signals. Immunohistochemistry and immunofluorescence are powerful techniques for detecting specific antigens, but they require optimal antibody selection, dilution, incubation, and detection. Other factors to consider are the staining time, temperature, and pH, which should be adjusted for each sample.

4 Ensure proper mounting and storage

The mounting and storage steps are important for preserving the quality and stability of the specimen. They protect the specimen from physical damage, fading, and deterioration. The mounting medium should match the refractive index of the specimen and the imaging objective. It should also prevent photobleaching, quenching, or autofluorescence of the fluorescent dyes or probes. Glycerol, polyvinyl alcohol, and antifade reagents are some of the common mounting media for fluorescence microscopy. The storage conditions should be appropriate for the type of specimen, the staining method, and the imaging modality. For example, paraffin sections can be stored at room temperature, but frozen sections should be stored at -20°C or -80°C.

Sample preparation for imaging studies is a complex and variable process that requires careful planning, optimization, and validation. By following these tips and best practices, you can improve your sample preparation and achieve better results and insights from your imaging studies.

5 Here’s what else to consider

This is a space to share examples, stories, or insights that don’t fit into any of the previous sections. What else would you like to add?