Choosing stem and epithelial marker antibodies: why an lgr5 antibody is famously hard to make work, what a muc2 antibody, a nectin-4 antibody and an endoglin antibody each report, and when a reporter line answers the question an antibody cannot

Choosing, and knowing when not to

1. Accept that some receptors have no reliable antibody. Low abundance surface receptors on rare cell populations are the hardest antibody targets there are, and several widely cited ones have a poor track record in independent hands. Before committing a study, search for independent validation rather than catalogue images, and treat a plausible pattern as a hypothesis.
2. Use a reporter line where the antibody is unreliable. A knock-in reporter at the endogenous locus reports expression without a reagent at all, and for the hardest of these targets it is the standard the field actually uses. It costs an editing project and it is the honest route when the antibody question has no good answer.
3. Match the preparation to a secreted mucin. Heavily glycosylated secreted proteins run as smears, are lost in some fixations and are extracted poorly by standard lysis. Expect to test fixation and extraction rather than assuming a protocol carries over from a cytoplasmic protein.
4. Check the epitope region for surface work. For a surface target being stained on intact cells or used to sort them, the epitope must be extracellular. Many catalogue reagents against these targets are raised against intracellular regions, which is stated on the datasheet and is the commonest reason a sort fails.
5. Validate on tissue where the expected pattern is known. These markers have well described distributions in normal tissue, which gives a built-in control: the right cells in the right place is evidence, and a uniform stain across a tissue that should be restricted is not.

Reading the independent literature rather than the catalogue

For a contested target, papers that used a genetic control alongside the antibody are worth far more than any number of catalogue images. Antibody validation databases and the methods sections of careful papers are the practical place to look.

Where a reagent has been used widely and never with a knockout control, that is information in itself. Widespread use is not validation, and for these targets it has repeatedly not been.

Transcript against protein

For low abundance receptors, in situ transcript detection is frequently more reliable than immunostaining and gives single cell resolution in tissue context. It answers a different question, and it is often the question that was actually being asked.

Where both are available, agreement between transcript and protein in the same tissue is strong evidence and disagreement is worth investigating rather than explaining away.

Buying prudently

Ask for a small trial size and test it against a genetic negative before committing a cohort. Suppliers confident in a reagent for a hard target will usually agree, and a refusal tells you something.

Record the catalogue number, lot and the validation you did. For contested targets your own record is what a reviewer will ask for, and it is what lets the next person in the laboratory avoid repeating the work.

Common questions

Why is an lgr5 antibody so hard to use?

The protein is at low abundance on a small population, which is the hardest combination there is. Several widely sold reagents have a poor record in independent hands, and a plausible-looking pattern is very hard to disprove without a genetic control.

What should I use instead?

A knock-in reporter at the endogenous locus, which reports expression without a reagent, or a transcript-level readout. For the hardest targets this is what the field relies on rather than staining.

Why does my muc2 antibody give a smear?

Heavy glycosylation and secretion. Mucins run broadly and high, are lost in some fixations and extract poorly under standard lysis, so both the fixation and the extraction need testing rather than inheriting.

Can I sort cells with these antibodies?

Only with an extracellular epitope and a validated live-cell protocol. Check the immunogen region first, because many reagents for these targets are against intracellular sequences and cannot stain an intact cell.

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