Fixation & processing
Immersion of tissue slices in 10–20 volumes of neutral buffered formalin for about 24–48 h, long enough for formaldehyde to reach and cross-link the centre but short enough to limit epitope masking.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Formalin fixation
Immersion of tissue slices in 10–20 volumes of neutral buffered formalin for about 24–48 h, long enough for formaldehyde to reach and cross-link the centre but short enough to limit epitope masking.
How it differs from the standard method
- Slice to 3–5 mm before fixing.
- Perfusion through the heart fixes whole organs from the inside in small animals.
- Record the time in fixative: it matters for immunostaining.
1Stopping the clock, then making tissue cuttable
The moment tissue loses its blood supply its own enzymes start to digest it. Fixation stops that: formaldehyde forms methylene bridges between proteins, locking them in place so the tissue keeps its structure through everything that follows.
2Penetration is fast, fixation is slow
Formaldehyde diffuses into tissue so that the distance it has travelled grows with the square root of time, d = K·√t, with K close to 0.8 mm per square-root hour. A 4 mm slice is reached at its centre (2 mm in) after about 7 hours; a 10 mm lobe needs nearly two days.
Under-fixation
The centre is finished off by the processor's alcohol instead of formalin. Cells shrink, nuclei smudge, and the centre stains differently from the edge (zonal fixation).
Over-fixation
Morphology looks perfect, but extra cross-links bury epitopes. Immunostaining weakens and needs harsher antigen retrieval.
Ischaemia and autolysis
Minutes on the bench cost phospho-epitopes and RNA; hours cost morphology.
Formalin pigment
Unbuffered formalin turns acidic and deposits brown-black haematin granules over blood.
3Dehydration, clearing and wax
Wax and water do not mix, so processing runs in steps. Graded ethanol baths remove the water gradually, a clearing agent such as xylene replaces the ethanol (wax does not mix with alcohol either), and molten wax replaces the xylene.
4Freezing without ice damage
For frozen sections the enemy is ice. Water that freezes slowly forms large crystals that push cells apart and leave holes in every section.
Key terms
Practise these as flashcards →10% formalin (about 4% formaldehyde) in phosphate buffer at pH 7; the standard histology fixative.
The constant in d = K·√t describing how far a fixative diffuses in a given time; about 0.8 mm/√h for formaldehyde.
Time between removing the tissue and getting it into fixative; ideally minutes.
A well-fixed rim around a core that formalin did not fix in time, with visibly different morphology and staining.
Replacing ethanol with a solvent miscible with wax, such as xylene; tissue turns translucent.
Replacing the clearing agent with molten wax so the whole tissue becomes solid and cuttable.
A water-soluble glycol and resin embedding medium that supports tissue for frozen sectioning.
Freezing in seconds, usually in isopentane chilled by liquid nitrogen, to keep ice crystals small.
When to use it
- Formalin fixation and paraffin embedding: routine morphology, H&E, most special stains and immunohistochemistry, and archiving blocks for years.
- OCT cryo-embedding: lipids (Oil Red O), enzyme histochemistry, epitopes destroyed by formalin or heat, and rapid diagnosis on the same day.
- Fixing a slice of a known thickness for a known time: whenever immunostaining will be scored or compared across samples.
Compared with alternatives
The next step: cutting the block on a microtome or cryostat, where a poorly processed block shows itself.
The routine read-out of fixation quality: zonal fixation, autolysis and pigment are all visible on H&E.
Most sensitive to fixation time: over-fixation masks epitopes and under-fixation gives uneven staining.
On cultured cells, fixation takes minutes because there is no tissue depth to penetrate.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Immersion of tissue slices in 10–20 volumes of neutral buffered formalin for about 24–48 h, long enough for formaldehyde to reach and cross-link the centre but short enough to limit epitope masking.
- Slice to 3–5 mm before fixing.
- Perfusion through the heart fixes whole organs from the inside in small animals.
- Record the time in fixative: it matters for immunostaining.