Fixation and Tissue Processing
What fixation must accomplish
Fixation stops autolysis and putrefaction while preserving morphology and, increasingly, antigenicity. 10% neutral buffered formalin is the default: it is only about 3.7–4% formaldehyde in phosphate buffer at pH 7.0–7.4, which prevents the acid-formalin (acid hematin) pigment seen with unbuffered formalin. Cross-linking occurs through methylene bridges between proteins. Use a 10:1 fixative-to-tissue volume ratio, blocks no thicker than 4 mm, and remember penetration of roughly 1 mm per hour - a 4 mm slice needs about 2 hours just to be penetrated, though routine practice is 6–24 hours.
Troubleshooting traps
Underfixation gives pale, mushy tissue with central autolysis; overfixation hardens tissue, causes shrinkage and cracking at microtomy, and masks epitopes for immunohistochemistry. Formalin pigment is brown-black and birefringent and is removed with saturated alcoholic picric acid, not by more washing. Bouin's fluid yellows tissue (remove with lithium carbonate or 70% alcohol) and Zenker's/B-5 leaves mercury pigment (remove with iodine).
Other fixatives and processing
Glutaraldehyde and osmium tetroxide serve electron microscopy (osmium preserves lipid); absolute alcohol is used for glycogen, which aqueous fixatives dissolve away; EDTA gives gentle decalcification that preserves antigen, whereas nitric or hydrochloric acid are fast but destructive. In processing, graded alcohols dehydrate, xylene clears, and paraffin infiltrates at 56–60 °C. Incomplete dehydration or clearing yields a milky block with bubbles.
Rule of thumb: if a block crumbles and looks like cheese, suspect incomplete fixation or processing before blaming the microtome.