Marker antibodies for tissue immunohistochemistry: what a cd31 antibody, a gfap antibody bought as an anti gfap antibody among the gfap antibodies sold for it, a p40 antibody used as a lineage marker and an iba1 antibody each label, why antibody markers for tissue need their own retrieval, where antibody blocking removes the background an immunohistochemistry antibody produces, and the controls a section needs

Getting a marker stain to work

1. Know what the marker labels, and what it does not. CD31 labels endothelium and is used to read vessel density, and it is also present on platelets and some leukocytes. GFAP labels astrocytes and rises with reactive change rather than with cell number. Iba1 labels microglia and macrophages and does not distinguish resident from infiltrating cells. Each of these caveats has been over-read in print.
2. Fix the fixation before blaming the antibody. Formalin fixation time changes epitope availability substantially, and over-fixed archival material needs harder retrieval. Record fixation time with the block; a cohort fixed inconsistently cannot be compared however good the staining looks.
3. Choose the retrieval condition empirically. Heat induced retrieval in citrate near pH six and in a Tris or EDTA buffer near pH nine give different results for the same antibody. Test both on a known positive section rather than following the first protocol you find.
4. Block what actually causes the background. Endogenous peroxidase in blood-rich tissue, endogenous biotin in liver and kidney, and non-specific protein binding each need a different block. Adding more serum does not fix a peroxidase problem.
5. Run the controls on the same slide run, every time. A known positive tissue, a negative tissue and a no-primary control. Where a section is scored quantitatively, the positive control also fixes the exposure and threshold, and without it two runs are not comparable.

Multiplexing three markers on one section

Raising the three primaries in different host species is the simplest route, because ordinary cross-adsorbed secondaries then separate them. Where two primaries share a host, sequential detection with an intervening blocking or stripping step is the alternative and it needs its own controls.

Choose fluorophores against the tissue's autofluorescence rather than against convenience. Brain and fixed archival tissue both fluoresce strongly in the green, which is where the brightest and cheapest conjugates sit.

Quantifying an image honestly

Fix the acquisition settings from the positive control and do not change them between sections. Auto-exposure across a cohort produces a dataset in which every section looks equally stained, which is the opposite of the measurement.

Decide the analysis before looking at the images: the region, the threshold rule and the unit. Thresholds chosen after seeing the result are the commonest source of an effect that will not replicate.

Buying the reagent

Prefer a clone with published images in your tissue and your fixation. For polyclonal reagents, buy a single lot for the whole cohort and record the lot with the data.

Recombinant monoclonals cost more and remove lot variation entirely. For a marker you will use across a multi-year study, that is usually the cheaper decision by the end.

Common questions

What does a cd31 antibody stain?

Endothelial cells, which is why it is the usual marker for vessel density. It also marks platelets and subsets of leukocytes, so a count based on it should be restricted to structures that look like vessels.

Does a gfap antibody count astrocytes?

Not reliably. It labels the intermediate filament, which is strongly upregulated in reactive astrocytes, so a stronger signal usually means more reactive cells rather than more cells. A nuclear marker is needed alongside it for counting.

What is an iba1 antibody used for?

Labelling microglia and macrophages, including their fine processes, which makes it the standard marker for microglial morphology. It does not separate resident microglia from infiltrating monocytes on its own.

Which retrieval buffer should I start with?

Try citrate near pH six and a Tris-EDTA buffer near pH nine on the same known positive tissue. Datasheet recommendations are a starting point and often reflect one laboratory's fixation rather than yours.

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