S100B antibody and glial and neural identity markers that survive activation

Building an identity panel

1. Pair a state independent marker with a state sensitive one. Counting cells needs a marker that does not change with activation. Reporting state needs one that does. Using a single marker for both jobs is how apparent cell loss is reported where there was a phenotype change.
2. Optimise retrieval per reagent. Nuclear transcription factors in fixed tissue frequently need retrieval to be visible, and the optimum differs between reagents. Optimise on tissue known to be positive rather than on the experimental sections.
3. Check the family, not just the member. Several of these targets belong to families whose members share sequence and are expressed in other cell types. Read the immunogen and check the datasheet for family cross reactivity before assigning a cell type.
4. Use morphology as a control on the marker. In tissue, the shape and position of a stained cell is independent evidence. A marker signal in cells with the wrong morphology is a warning, not a discovery.
5. Count with a defined rule and a blinded scorer. Cell counting in sections is dominated by sampling and by scoring rules. Define the region, the section interval and the inclusion criteria in advance, and score blind.

Identity and state are entangled

The markers used to name cells in nervous tissue were mostly discovered because they are strongly expressed, and strong expression is frequently regulated. That entanglement is the central difficulty of the field and it is not solved by a better reagent.

It is solved by panels: one marker chosen for stability, one for state, and morphology as a third line of evidence.

Sections are sampled, not measured

A count from a section is an estimate that depends on section thickness, interval, region definition and how partial cells are treated. Two studies reporting different numbers frequently differ in sampling rather than in biology.

State the sampling scheme with the result. It is what allows the numbers to be compared with anyone else's.

An rbpms antibody and the retinal ganglion cell

RBPMS labels retinal ganglion cells specifically, which is why an rbpms antibody has become the standard reagent for counting them in injury and glaucoma models, and the readout is cells per area with a stated sampling rule. It is a nuclear and cytoplasmic RNA binding protein, so the pattern is not purely nuclear, and a retinal layer position is part of the identification.

A pgp 9.5 antibody as a pan neuronal marker

PGP 9.5 is a ubiquitin hydrolase abundant in neurons, and a pgp 9.5 antibody is used to show nerve fibres in tissue where a more specific marker would miss most of them, notably in intraepidermal fibre counting. Because it is abundant, the failure mode is background rather than sensitivity, and a stated fibre counting rule is what makes the number comparable.

A serotonin antibody and a small molecule epitope

Serotonin is a small molecule, so an antibody against it is raised on a carrier conjugate and the fixation has to crosslink the transmitter in place: a serotonin antibody used after a routine formaldehyde protocol may find nothing because the target washed out. Perfusion fixation and the vendor's own protocol matter more here than the clone.

An aldh1l1 antibody and an astrocyte marker beside the page's own

ALDH1L1 labels astrocytes more completely than the classical marker, which stains only a subset strongly, so an aldh1l1 antibody is chosen when the question is how many astrocytes rather than how reactive they are. The two markers disagree by design, and a figure claiming astrocyte number should say which was used. It is cytoplasmic and fills processes.

A tmem119 antibody and microglia against infiltrating cells

TMEM119 distinguishes resident microglia from infiltrating monocytes, which the shared myeloid markers cannot do, so a tmem119 antibody is the reagent when the origin of a cell is the question. Expression falls as microglia activate, which means a negative cell in an inflamed tissue may be an activated resident rather than an infiltrate, and that caveat belongs in the figure.

An rbfox3 antibody and the marker sold as NeuN

The neuronal nuclear antigen known as NeuN is RBFOX3, so an rbfox3 antibody and a NeuN antibody may be the same reagent under two names and searching one hides the other. It is nuclear with some cytoplasmic signal, and several neuron types express it weakly or not at all, so a count based on it undercounts by a known amount that belongs in the method.

An nse antibody and neuron specific enolase in two uses

Neuron specific enolase is read in tissue as a neuroendocrine marker and in serum as a tumour or injury marker, so an nse antibody in a catalogue may be for either. Haemolysis releases the enzyme from red cells and inflates a serum figure, which makes sample handling the dominant variable. For a section, the expected negative populations are what make the stain interpretable.

Common questions

Does a reduced marker mean cell loss?

Not on its own. Activation, injury and maturation all change marker expression, so a second marker with different regulation, or a nuclear count, is what distinguishes loss from a state change.

Why is my transcription factor stain weak?

Retrieval, usually. Nuclear targets in cross linked tissue need conditions optimised per reagent, and a weak nuclear stain is far more often a retrieval problem than a reagent problem.

Can one marker such as a DCX antibody define a glial population?

Rarely. Family members are shared with other cell types and expression shifts with state. Two markers plus morphology is the practical minimum for a population claim.

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