Preparing epithelial tissue for transmission electron microscopy (TEM) involves a series of critical steps to preserve the tissue's ultrastructure and provide sufficient contrast for imaging. Here's a detailed overview of the process:
1. Fixation: Preserving the Tissue
The initial step is fixation, which stabilizes the tissue's proteins and lipids, preventing degradation and distortion during subsequent processing.
1.1. Primary Fixation (Aldehydes)
- Purpose: Primarily targets and cross-links proteins.
- Common Fixatives: Glutaraldehyde (2-4%) and/or formaldehyde (4%) in a buffer solution (e.g., phosphate buffer, cacodylate buffer).
- Procedure: Immerse the epithelial tissue sample in the aldehyde fixative for a period ranging from a few hours to overnight, depending on the tissue type and size. Perform at 4°C to slow down enzymatic activity.
- Example: 2.5% glutaraldehyde in 0.1M phosphate buffer, pH 7.4, for 2 hours at 4°C.
1.2. Secondary Fixation (Osmium Tetroxide)
- Purpose: Preserves and stabilizes lipids, adding contrast to membranes.
- Fixative: Osmium tetroxide (OsO4), typically a 1-2% solution in the same buffer as the primary fixative.
- Procedure: After rinsing the tissue to remove residual aldehyde, immerse it in the osmium tetroxide solution for 1-2 hours. Osmium tetroxide is a hazardous material and must be handled with appropriate safety precautions (fume hood, gloves, etc.).
- Example: 1% OsO4 in 0.1M phosphate buffer, pH 7.4, for 1 hour at room temperature.
2. Staining (Optional)
2.1. Tertiary Fixation and Contrasting (Uranyl Acetate)
- Purpose: To increase contrast by binding heavy metals to cellular structures.
- Stain: Uranyl acetate, a radioactive heavy metal salt, enhances contrast in the TEM image.
- Procedure: Incubate the sample in a saturated aqueous solution of uranyl acetate for 30 minutes to 1 hour in the dark.
- Example: Saturated uranyl acetate solution for 1 hour in the dark.
3. Dehydration: Removing Water
Water interferes with the resin embedding process. Therefore, dehydration is essential.
- Procedure: Gradually replace water with a solvent (typically ethanol or acetone) through a series of increasing concentrations. This prevents excessive shrinkage or distortion.
- Dehydration Series Example:
- 30% ethanol (or acetone) for 10 minutes
- 50% ethanol (or acetone) for 10 minutes
- 70% ethanol (or acetone) for 10 minutes
- 90% ethanol (or acetone) for 10 minutes
- 100% ethanol (or acetone) for 2 x 15 minutes (anhydrous)
4. Infiltration and Embedding: Providing Support
The tissue needs to be embedded in a solid resin to allow for ultra-thin sectioning.
- Procedure: Gradually infiltrate the dehydrated tissue with a resin that will polymerize to form a hard block.
- Infiltration: Replace the dehydrating solvent with increasing concentrations of resin in solvent (e.g., 25%, 50%, 75%, 100% resin in ethanol/acetone). This ensures the resin fully penetrates the tissue. The infiltration steps may take several hours or overnight.
- Embedding: Place the infiltrated tissue in a mold filled with fresh resin and polymerize the resin by heating according to the manufacturer's instructions. Common resins include epoxy resins (e.g., Epon, Araldite) or acrylic resins.
5. Sectioning: Creating Ultra-Thin Slices
Ultra-thin sections are required for TEM imaging.
- Procedure: Use an ultramicrotome equipped with a diamond knife to cut sections approximately 50-100 nm thick.
- Floating: Sections are floated onto a water bath in the ultramicrotome to allow them to spread and flatten.
6. Mounting and Staining (Post-Staining)
- Mounting: Carefully pick up the sections from the water surface with copper or nickel grids.
- Post-Staining (Optional): Enhance contrast further by staining the sections on the grids with heavy metal stains, such as uranyl acetate and lead citrate. This is commonly done to enhance contrast of structures like membranes and ribosomes.
7. Imaging: Observing the Ultrastructure
The prepared sample is now ready for examination using a transmission electron microscope.
