You may ask Michele Miragoli: [email protected]

or

Domenico Corradi: [email protected]

sorry: [email protected]

Creating histological sections of the sinoatrial node (SAN) of the heart requires careful preparation due to the delicate and specific nature of the tissue. Here’s a step-by-step guide to help you:

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1. Tissue Collection

Obtain the heart: Ensure you have ethical and proper approval for using animal or human tissue, depending on your study.

Locate the SAN: The SAN is situated at the junction of the superior vena cava (SVC) and the right atrium. Carefully dissect this region to include the SAN and surrounding tissue.

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2. Fixation

Fix the tissue: Immediately immerse the dissected region in an appropriate fixative to preserve the tissue morphology. Common fixatives include:

10% Neutral Buffered Formalin: Good for general histology.

Paraformaldehyde (PFA) 4%: Ideal for immunohistochemistry.

Fixation time will depend on the tissue size, typically 12–24 hours at 4°C.

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3. Embedding

Dehydrate the tissue: Process the tissue through a graded ethanol series (70%, 80%, 90%, 95%, and 100%) to remove water.

Clear the tissue: Use a clearing agent like xylene to make the tissue compatible with the embedding medium.

Embed in paraffin: Infiltrate the tissue with melted paraffin wax, then embed it in a mold, ensuring proper orientation.

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4. Sectioning

Cut thin sections: Use a microtome to slice 5–10 µm thick sections of the paraffin-embedded tissue.

Place on slides: Float the sections on a water bath (around 40–45°C) and transfer them to glass slides coated with adhesive (e.g., poly-L-lysine) to ensure they stick during staining.

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5. Staining

Hematoxylin and Eosin (H&E): Use for general morphology to differentiate nuclei and cytoplasm.

Special stains: To identify SAN-specific features:

Masson's Trichrome: Highlights connective tissue.

Elastic stains: Visualize elastic fibers if needed.

Immunohistochemistry (IHC): Use antibodies specific to SAN markers like HCN4, connexin-43, or Tbx3 for precise identification of SAN tissue.

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6. Microscopy and Imaging

Use a light microscope to examine the stained sections.

Capture images using a camera attachment for analysis and documentation.

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Additional Tips

Proper orientation: Ensure you section the SAN along the desired plane (e.g., longitudinal or transverse) to study specific structural details.

Consult an atlas: Use anatomical atlases or references to confirm the precise location of the SAN.

Cryosectioning (optional): If fresh tissue is required for molecular studies, you can embed it in OCT compound and cut sections using a cryostat instead of paraffin embedding.

Would you like more details on any specific step?