Fixation, Processing, Embedding, and Microtomy
Fixation
The default fixative is 10% neutral buffered formalin (NBF), a cross-linking fixative whose active species is formaldehyde; the buffer holds pH at 7.2-7.4, where cross-linking is optimal and formalin pigment (acid formaldehyde hematin) is minimized. Use a 10:1 fixative-to-tissue volume ratio and allow roughly 1 mm of penetration per hour, so a 5 mm slice needs at least 5-6 hours and routine small biopsies 6-24 hours. Overfixation (beyond 48-72 hours) masks epitopes and hardens tissue; underfixation produces pale staining, streaming artifact and poor morphology. Coagulant fixatives (Bouin, Zenker, B5, Carnoy) have specific uses but may require washing or treatment to remove pigment, and several damage DNA.
Processing and embedding
Processing runs graded ethanol dehydration, xylene clearing, then paraffin infiltration. Traps: incomplete dehydration leaves milky xylene and brittle blocks; over-dehydration shrinks and hardens tissue; incomplete clearing leaves alcohol in the block, so ribbons crumble. Embedding orientation matters: orient skin, mucosa and muscle so the diagnostic plane is cut first.
Microtomy
Routine sections are 3-5 µm (4 µm typical; 2-3 µm for IHC). Chatter (venetian-blind artifact) comes from a loose blade, hard paraffin or a dull edge; alternating thick-thin sections suggest vibration or a loose block. Acid decalcifiers (nitric, formic) are fast but destroy DNA and many epitopes; EDTA is slow and gentle.
Rule of thumb: 10:1 volume, pH 7.2-7.4, about 1 mm per hour, 4 µm sections. If a block shatters, suspect processing before you blame the microtome.