Fig 1: Summary of immunocytochemical staining of PPGL tumour cultures.Both chromogranin A (Tu8_Chromogranin A_10x obj.) and neuron-specific enolase (Tu-28_Neuron-specific enolase (NSE)_10x obj.) are generally negative, even in very early cultures. Tyrosine hydroxylase staining (Tu36_Tyrosine hydroxylase (TH)_40x obj.) is often positive, even in long-term cultures, but as many HNPGL cultures are negative for TH at the outset this marker is not generally applicable. In our hands the most broadly reliable marker in PPGL tumour cultures proved to be synaptophysin (Tu18_Syn_40x obj.), as this marker can be found in both early and late cultures. To assess the survival of neuronal cells in PPGL tumour cell cultures we also stained for neurofilament protein (Tu-7_Neurofilament protein_20x obj.), which is expressed in nerve fibres and the sparse individual neurons seen in PPGL tumours. We found that even at 29 months of culture (Tu7), a few cells of neuronal morphology are still present in culture. CD56 (NCAM) is a cell surface marker for chromaffin cells and is found in most PPGLs, showing the distinct staining pattern of a cell surface protein. Of the few tumour cultures examined (Tu-42_CD56 (ab 123C3) _10x obj.), only sparse cells were positive for CD56. The classic markers for sustentacular cells in PPGLs are S100 and GFAP. S100 tends to show more widespread staining of these cells and is therefore favoured in IHC applications, while GFAP is expressed more sporadically in the same cells. Both marker proteins are expressed in tumour cultures but S100 expression (Tu-40_S100_20x obj.) appears to be short-lived, while GFAP expression (Tu44_thoracic PGL_GFAP_20x obj.) persists somewhat longer, both generally disappearing over the course of 4–8 weeks, along with cells showing sustentacular morphology. CD31 is a marker for endothelial cells and staining of PPGL FFPE tumour sections underscores the extensive vascularity of these tumours. Although few cultures were stained with CD31, those that were appeared uniformly negative (Tu-8_CD31_20x obj.), suggesting that endothelial cells do not persist in culture. Ki-67, a protein expressed in proliferating cells, was occasionally positive in cells of fibroblast morphology (Tu-52_Ki-67_20x obj.). Cytokeratin, a marker for epithelial cells, was occasionally positive (Tu-8_Cytokeratin_10x obj.) in a few cells. Cytoskeleton proteins such as smooth muscle actin (SMA), vimentin and fibronectin are present in most cells and as such are aspecific markers. However, they can usefully illustrate the general morphology of cell cultures. We found SMA (Tu-47_Smooth muscle actin (SMA)_40x obj._cytospin) and vimentin (Tu-28_Vimentin_10x obj.;Tu-8_Vimentin_10x obj.) to be the most useful, with fibronectin only sporadically positive in a few cells (Tu-17 _Fibronectin_20x obj.).