High-quality FFPE tissue sections are the cornerstone of accurate and reliable data in spatial omics workflows, especially when it comes to GeoMx DSP and CosMx SMI. Think of it like building a house: a solid foundation is essential. In this case, that foundation is meticulous tissue handling and preparation. This ensures those precious biomolecules stay intact and your assays perform at their best. So, let’s dive into the best practices for getting your FFPE tissues ready for GeoMx and CosMx.
Pre-Analytical Phase: Setting the Stage for Success
The journey begins with the pre-analytical phase. This covers everything from the moment you collect the tissue to when it’s fixed and processed. Don’t underestimate this stage – it has a huge impact on the quality of your spatial omics data down the line.
- Minimize Ischemia Time: Time is of the essence! The quicker you get that tissue from the body into fixative, the better. Think of it as a race against time to preserve those RNA and protein molecules. Ideally, you want to plunge that tissue into fixative immediately.
- Optimal Fixation: A Balancing Act
- Fixative Choice: Stick with the gold standard: 10% Neutral Buffered Formalin (NBF). It’s the recommended fixative for both GeoMx and CosMx.
- Fixation Time: Finding the Sweet Spot: Aim for a fixation time between 6 and 24 hours. Overdoing it can cause those nucleic acids and proteins to crosslink (not good!), while under-fixation can lead to degradation. And whatever you do, avoid letting that tissue sit in fixative for too long (over 48 hours).
- pH Matters: Keep that formalin solution neutral (pH 7.0-7.4). This helps prevent those precious biomolecules from breaking down.
- Tissue Dimensions: Thinner is better! Aim for tissue samples no thicker than 5 mm. This ensures the fixative can penetrate effectively, like a marinade soaking into a steak.
- Prompt Processing: Once the tissue is fixed, don’t let it sit around. Process it as quickly as possible to minimize degradation.
Tissue Processing and Embedding: Treat Your Tissues with TLC
Now it’s time to process and embed those tissues. Remember, consistency is key!
- Automated Processing: Use an automated tissue processor with a validated protocol. This ensures consistent and controlled processing, like a well-oiled machine.
- Minimize Processing Time and Temperature: While you need to ensure complete dehydration and clearing, try to keep processing times and temperatures as low as possible. Heat and harsh solvents can be tough on those delicate biomolecules.
- Solvent Considerations: If feasible, consider using gentler alternatives to ethanol and xylene. It’s like choosing a milder soap for a delicate garment.
- Low Melting Point Paraffin: Use paraffin with a low melting point (56-58°C) to minimize heat-induced damage.
- Optimal Embedding: Embed the tissue carefully, making sure your region of interest is positioned correctly for sectioning. And watch out for those pesky air bubbles!
Sectioning: Precision is Key
Time to get slicing!
- Section Thickness: For GeoMx and CosMx, a 5 µm section thickness is just right. It allows for optimal reagent penetration and keeps background noise to a minimum.
- Microtome Maintenance: A well-maintained microtome with a sharp, new blade for each block is essential. This ensures consistent section thickness and minimizes tissue damage. Think of it like using a sharp knife to slice bread – you get clean, even slices.
- RNase-Free Environment (For RNA Analysis): If you’re working with RNA, remember that RNases are everywhere, just waiting to degrade your samples. Maintain an RNase-free environment by cleaning your microtome and work area with RNase decontamination solutions and wearing gloves.
- Slide Mounting: Use positively charged slides (like Leica Bond Plus or SuperFrost Plus) for optimal tissue adhesion.
- Baking Sections: Baking those sections at 60°C for 30-60 minutes can help with tissue adhesion and reduce background. However, it’s best to optimize the baking time based on your tissue type and assay. Detachment-prone tissues need a little extra attention here.
- Section Storage: Freshly cut sections are always best. But if you need to store them, keep them in a dry, dark place at room temperature for up to a few weeks. Be sure to check the Nanostring protocols for specific storage recommendations.
GeoMx DSP: Specific Considerations
- Region of Interest (ROI) Selection: Choose your ROIs wisely! Consider tissue morphology, cellular heterogeneity, and your research goals.
- Decrosslinking and Antigen Retrieval: Follow NanoString’s recommended protocols for these steps, tailored to the specific antibodies you’re using.
CosMx SMI: Specific Considerations
- Tissue Permeabilization: Make sure those probes can penetrate effectively by following NanoString’s recommended permeabilization protocols.
- Image Acquisition and Analysis: Stick to NanoString’s guidelines to ensure accurate and reliable data.
RNA Preservation: Handle with Care
Whether you’re working with GeoMx or CosMx, if RNA is involved, treat it like a precious gem!
- RNase Inhibitors: If you’re using an ice bath rehydration method, don’t forget to include an RNase inhibitor.
- Minimize Handling: The less you handle those sections, the better. Every touch increases the risk of RNase contamination.
- RNase-Free Reagents and Consumables: Make sure everything you use is RNase-free.
Quality Control: The Final Check
- Tissue Quality Assessment: Before you go any further, take a look at your FFPE tissue sections under the microscope with an H&E stain. Make sure the morphology looks good and there’s minimal damage.
- Reagent Quality Control: Always use fresh, high-quality reagents and consumables.
- Instrument Calibration and Maintenance: Keep your instruments calibrated and well-maintained for optimal performance.
- Manufacturer’s Recommendations: NanoString knows their stuff! Adhere to their recommended protocols and guidelines.
By following these tips, you’ll be well on your way to generating high-quality data from your GeoMx and CosMx experiments. Good luck!
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