Fig 1.
PTA block staining of human and murine skin tissue and human lung parenchyma.
Labelling. White asterisks: transversal sections of collagen I/III fibrils, black asterisks: longitudinal sections of collagen I/III fibrils, white arrows: keratin fibres, black arrows: desmosomes, white arrowheads: hemidesmosomes, black arrowheads: collagen VII anchoring fibrils, white pentagon arrows: collagen I/II fibril profiles showing inhomogeneous staining patterns. af: anchoring filaments bm: basal membrane, E: endothelial cell, ef: elastic fibres, er: endoplasmic reticulum, g: Golgi apparatus, ld: lamina densa, ll: lamina lucida, m: mitochondrion, n: nucleus, P1: type 1 pneumocyte, P2: type 2 pneumocyte, sv: surfactant vesicle. (a) Healthy human skin, overview of the dermoepidermal junction. PTA prominently stains the keratin fibres and desmosomal and hemidesmosomal plaques of the keratinocytes. Aggregates of collagen I/III fibrils and the lamina lucida and lamina densa sublayers are clearly visible. (b) Healthy murine dermis. Collagen I/III fibrils are stained at high contrast, with clearly visible banding in longitudinal stretches. (c) Human in-vitro skin equivalent of a healthy control donor, dermoepidermal junction. E-PTA-staining accentuates the keratin fibres connecting to the hemidesmosomes. Anchoring filaments traversing the lamina lucida and collagen VII anchoring fibrils connecting the basal lamina to the dermal collagen are clearly visible. Again, the banding of longitudinally sectioned collagen I/III fibrils is well defined. Transversal profiles of these fibrils frequently show inhomogeneous staining patterns. (d,e,f) Fibrotic human lung parenchyma. (d) Low magnification overview. E-PTA staining allows for the differentiation of all cellular and extracellular components and particularly accentuates the elastic fibres. (e) Detail of an alveolar septum. Large deposits of collagen are clearly visible, pneumocytes and endothelial cells are well defined. (f) Detail of an alveolar fibroblast demonstrating the suitability of E-PTA for staining of intracellular components. Mitochondria, Golgi apparatus and endoplasmic reticulum are clearly defined, ribosomes and nuclear chromatin are especially well stained. Scale bars: (a-c): 0.5 µm, (d-f): 1.0 µm.
Fig 2.
Comparison of staining methods on human skin equivalents.
(a-c): Section staining using UA replacement stain and lead citrate (alternative 1). (d-f): Combination of E-PTA and sections staining as above (alternative 2). (g-i): E-PTA block staining. Labelling. White asterisks: transversal sections of collagen I/III fibrils, black asterisks: longitudinal sections of collagen I/III fibrils, white arrow: keratin fibres, black arrows: desmosomes, white arrowheads: hemidesmosomes, white daggers: elastic fibrils. er = endoplasmic reticulum, K = keratinocyte, ld: lamina densa, ll: lamina lucida, m = mitochondrion, n = nucleus. Intracellular components are well defined with all staining methods (a,d,g). Ribosomes and inner membrane structures are slightly better visible with routine section staining alone (a,b), although the combination of E-PTA and section staining results in the best overall intracellular contrast (d,e). Keratine fibres are strongly stained by E-PTA (g,h), and this effect is further enhanced by additional section staining (d,e). The basal lamina at the dermoepidermal junction is visible with all staining methods (c,f,i). The potential of PTA block staining is best demonstrated by its effects on desmosomes (d,g) and hemidesmosomes (e,h), and by its superior staining of collagen fibrils. Routine section staining results in poor definition of collagen I/III fibrils, especially in cross sectional view (b,c). By contrast, E-PTA stained collagen I/III fibrils always exhibit high contrast and a clearly visible banding pattern (h,i). Additional section staining weakens E-PTA collagen contrast rather than improving it (e,f). Scale bars: 0.5 µm.