Reading tumour suppressors and oncogenes without reading the mutation wrongly: why an anti p53 antibody stains strongly in exactly the tumours where the protein is broken, because a stabilising mutation raises the level of a non functional protein, what a pten antibody and a vhl antibody report by absence and how hard absence is to prove with a stain, how a kras antibody, a kras g12d antibody and an hras antibody differ in whether the reagent distinguishes the mutant, where an idh1 antibody and an mtap antibody are used as mutation surrogates, what an nf1 antibody, a wt1 antibody, a runx1 antibody, an erg antibody, an etv4 antibody, a trps1 antibody, a pax5 antibody, a gata1 antibody, a gata6 antibody and a tcf4 antibody report as lineage rather than driver, and how a cmyc antibody, an n myc antibody and an n-myc antibody are read against amplification

Reading these stains correctly

1. Know which direction the stain runs. For proteins stabilised by mutation, high signal indicates a likely mutation and low signal indicates either normal turnover or a truncating change. The stain is a pattern, and the pattern has to be interpreted rather than read as amount.
2. Treat absence as the hardest claim. A negative stain can mean loss, a failed reagent, poor fixation or an epitope that was removed by a truncation. An internal positive control within the same section, such as normal stroma, is what makes absence credible.
3. Use mutation specific reagents only for their exact change. A reagent raised against one substitution detects that substitution and usually not its neighbours. Reporting it as detecting mutant protein generally is an overstatement.
4. Confirm surrogates against sequence. Where a stain is used as a proxy for a genotype, its concordance with sequencing has to be established in your own material before it is relied on.
5. Separate lineage from driver. Several reagents in this group identify a cell of origin rather than a driver event. Using them to infer mechanism is a common and avoidable overreach.

Accumulation is the signal, not the function

The stain reports steady state protein, and steady state is set by synthesis and degradation. A change that blocks degradation raises the level of a protein that no longer works, which is why the intuitive reading is unreliable here more than anywhere else.

Describe the pattern in the methods and the interpretation in the discussion, rather than reporting intensity as if it were activity.

Internal controls make sections interpretable

Almost every tissue section contains cells that should be positive: stroma, immune infiltrate, adjacent normal epithelium. Using them as internal controls turns a stain into a comparison rather than an absolute.

Point them out in the figure. A reader can then judge for themselves whether a negative tumour was truly negative or the section simply did not stain.

Common questions

Does strong staining mean the suppressor is working?

Often the opposite. Missense changes that abolish function also abolish the degradation that keeps the protein low, so accumulation is a marker of a likely mutation rather than of activity.

Can a stain replace sequencing?

For a few well characterised changes with validated reagents, it can be an effective screen. It is not equivalent, and the concordance has to be established locally before the stain is used in place of the sequence.

How do I make a negative stain credible?

With an internal positive control in the same section, a reagent whose epitope is unlikely to be lost by truncation, and where possible a second reagent against a different region.

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