Choosing a pcna antibody, a tubulin antibody or another nuclear marker antibody: what each one actually reports about a cell's state, why proliferation markers are not interchangeable, and the fixation that decides whether either stain works

What each marker reports

1. A proliferation marker reports recent history, not a snapshot. The protein accumulates through the cell cycle and persists after the cell leaves it, so staining reports cells that have cycled recently as well as those cycling now. Where the question is which cells are in S phase at this moment, a nucleotide incorporation assay answers it and an accumulation marker does not.
2. Choose the proliferation marker to match the question. Different proliferation antigens have different persistence and different cell cycle profiles, so they give different indices on the same tissue. Report which one was used and do not compare an index measured with one against a published figure measured with another.
3. Handle cytoskeletal staining as a fixation problem. Microtubules depolymerise in the cold and are poorly preserved by some fixatives. Aldehyde fixation at temperature, or a methanol protocol, each preserve different aspects, and a disappointing cytoskeletal image is nearly always fixation rather than antibody.
4. Anchor every count to a nuclear stain. A proliferation index is a fraction, so the denominator has to be counted. A general nuclear marker counterstain in a separate channel is what makes the fraction measurable rather than estimated, and it also exposes tissue folds and debris.
5. Run the controls that make the index credible. A tissue known to be proliferating, one known not to be, and a no-primary control on the same run at the same settings. An index reported without them is an impression with a number attached.

Quantifying an index honestly

Fix the acquisition settings from a control section and do not change them across a cohort, define the region and the threshold before looking at the images, and count enough cells that the fraction has a meaningful confidence interval.

Auto-exposure across a cohort makes every section look equally stained, which is the opposite of the measurement. This is the most common way a proliferation index stops being a measurement.

Cytoskeletal imaging beyond the stain

Post-translational modifications of tubulin mark stable against dynamic microtubules and are detected by separate antibodies, which is a considerably more informative experiment than total tubulin staining.

For live dynamics, a fluorescent protein fusion or a small molecule probe answers questions fixation cannot. A fixed image of a dynamic structure is a single frame of a film.

Buying the reagent

Prefer a clone with published images in your tissue and your fixation, and prefer a recombinant monoclonal for a study running over years. For polyclonal reagents, buy one lot for the whole cohort and record it.

Titrate on your own sections rather than using the datasheet dilution. Proliferation markers in particular are frequently used far too concentrated, which raises background and inflates the index.

Common questions

What does a pcna antibody actually show?

A protein that accumulates during the cell cycle and persists for a period afterwards, so it marks recently and currently cycling cells. It is an accumulation marker, not a snapshot of cells in S phase.

Are proliferation markers interchangeable?

No. They differ in cell cycle profile and in how long they persist, so the index they give on the same tissue differs. Always state which marker produced an index, and do not compare across markers.

Why does my tubulin antibody give a poor image?

Fixation, almost always. Microtubules depolymerise in the cold and some fixatives preserve them badly. Test an aldehyde protocol at temperature against a methanol protocol on a known positive before changing antibody.

Do I need a nuclear counterstain?

For anything quantitative, yes. A proliferation index is a fraction and the denominator has to be counted, which needs a general nuclear marker in its own channel. It also reveals folds and debris that would otherwise be counted.

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