Must I cut them immediately with a microtome or can I keep them and use them over time?
Storing plant samples in paraffin is a common technique used in histology to preserve tissues for later examination. Here’s a comprehensive guide on how long you can store these samples and best practices for handling them, including whether you need to cut them immediately with a microtome or can store them for future use.
Storing Plant Samples in Paraffin
1. Stability and Longevity:
- Long-Term Storage: Plant samples embedded in paraffin can be stored for a very long time, typically years, without significant degradation if stored under proper conditions. Paraffin provides a stable medium that protects the samples from environmental factors.
- Environmental Conditions: Store the paraffin-embedded samples in a cool, dry place, away from direct sunlight, to prevent any potential damage due to temperature fluctuations and humidity.
2. Storage Containers:
- Paraffin Blocks: Keep the paraffin blocks in airtight containers or sealed plastic bags to protect them from dust and moisture.
- Labeling: Ensure that each block is properly labeled with relevant information, such as the sample ID, date of embedding, and any other pertinent details.
Cutting with a Microtome
1. Immediate vs. Delayed Cutting:
- Immediate Cutting: You can cut the samples immediately after embedding them in paraffin. This is often done if immediate analysis is required.
- Delayed Cutting: It is entirely feasible to store the paraffin blocks and cut them at a later date. This flexibility allows researchers to plan their analysis according to their schedule and workload.
2. Handling Stored Paraffin Blocks:
- Re-Softening Paraffin: Before cutting stored paraffin blocks, you might need to re-soften the paraffin to make sectioning easier. This can be done by placing the blocks in a cool room or refrigerator for a short period before sectioning.
- Avoiding Brittleness: Over time, paraffin can become brittle. If you encounter this issue, gently warming the blocks (e.g., in a paraffin oven or warm water bath) can help restore the paraffin’s pliability.
Best Practices for Cutting and Staining
1. Preparing the Microtome:
- Sharp Blades: Ensure the microtome blades are sharp to obtain clean and consistent sections. Dull blades can cause tearing or compression of the tissue.
- Section Thickness: Adjust the microtome to cut sections at the desired thickness, typically between 5-10 micrometers for most histological studies.
2. Section Handling:
- Water Bath: Float the sections on a warm water bath (around 40-45°C) to flatten them before transferring them to slides. This step helps in reducing wrinkles and folds.
- Adhesion to Slides: Use adhesive-coated slides or apply an adhesive (e.g., poly-L-lysine) to standard slides to ensure the sections adhere properly during subsequent staining.
3. Staining:
- Timing: You can stain the sections immediately after cutting or store the slides and stain them later. If you choose to store unstained sections, keep them in a dust-free environment, ideally in slide boxes.
- Protocols: Follow the specific staining protocols suited for your study. Common stains for plant tissues include toluidine blue, safranin, and fast green.
Considerations for Specific Research Needs
1. Type of Plant Tissue:
- Different plant tissues (e.g., leaves, stems, roots) may have varying storage and cutting requirements. For example, woody tissues might be more challenging to section and may require more careful handling.
2. Research Objectives:
- Align your storage and cutting practices with your research objectives. For instance, if you are conducting a time-series analysis, ensure consistency in storage and handling practices to avoid introducing variability.
Summary
- Long-Term Storage: Paraffin-embedded plant samples can be stored for years if kept in a cool, dry place.
- Cutting Flexibility: You do not need to cut the samples immediately. They can be stored and sectioned at a later date.
- Handling Practices: Proper storage, re-softening techniques, and careful microtome preparation are crucial for maintaining sample integrity.
- Staining: Sections can be stained immediately or stored for future staining, depending on your research timeline.
By following these guidelines, you can effectively store and handle paraffin-embedded plant samples, ensuring their integrity for future analysis.
Paraffin-embedded plant samples can typically be stored under proper conditions for 1-5 years. However, some samples may remain usable for over a decade if well-prepared and maintained in a cool, dry environment.
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