Embedding and Microtomy
Orientation is the whole game
Embedding determines what the microtomist can deliver. The rule: the surface you want to examine first must face the bottom of the mold, because that is the surface the knife strikes first. Tubular structures such as vessels or bowel are embedded on edge for cross-sections, or longitudinally, but never rolled. Skin is embedded perpendicular to the epidermal surface so epidermis, dermis, and subcutis all appear in one section. Multiple fragments go in one plane without overlap.
Thickness and angles
Routine H&E sections are 3–5 µm (4 µm is standard), 2–3 µm for biopsies and kidney, 1–2 µm for research. The clearance angle between knife and block is typically 3–8° (about 5°). Too little clearance and the block face rubs, producing compression; too much and you get "venetian blind" or "picket fence" chatter.
Recognizing artifacts
Alternating thick and thin segments (chatter) come from vibration - a loose knife or block, too steep a clearance angle, or very hard tissue; slow the cut and tighten the clamp. Fine parallel longitudinal scratches indicate knife nicks or debris. Holes in the section usually mean incomplete fixation, incomplete processing, or bubbles in the paraffin. Wrinkles and folds are resolved on a water bath at 40–45 °C; too hot stretches and distorts the section, too cold leaves wrinkles. Curling suggests a dull blade. Frozen sections are cut near −20 to −25 °C in a cryostat; the block should be cold but not frosted white, and OCT should not be allowed to thaw.
Rule of thumb: check the trimmed block face - if the full cross-section of every piece is not visible, reorient before cutting a ribbon.