Fig 1: Double-label immunofluorescence of IBA1 and S100; and IBA1 and cytosolic ferritin in DRG of normal controls and FA. a-b normal controls; c-d FA. a and c double-label immunofluorescence of S100 (Alexa Fluor 488 green) and IBA1 (Cy3 red); DAPI (blue); b and d double-label immunofluorescence of IBA1 (Alexa Fluor 488 green) and cytosolic ferritin (Cy3 red). In the normal DRG, IBA1-reactive monocytes may gain access to the S100-reactive satellite cell sheath around neurons (N) but remain separated from the neuronal plasma membrane by a thin layer of satellite cell cytoplasm (a, arrow). IBA1 and S100 show no colocalization. In FA, the outline of the S100-positive satellite cell layer appears irregular and disrupted. An IBA1-reactive monocyte (arrow) abuts or penetrates the neuronal plasma membrane (c). The inset in (c) shows infiltration of a residual nodule by IBA1-positive monocytes. DAPI fluorescence in (c) confirms increased cellularity in FA when compared to the normal DRG (a). A normal DRG shows ferritin immunofluorescence in satellite cells and neurons (N) (b). The mixed yellow and green color suggests that ferritin biosynthesis also occurs in IBA1-positive monocytes. In FA, a multilayered rim of satellite cells around a shrunken neuron (N) is intensely fluorescent for cytosolic ferritin (Cy3 red) (d). Ameboid monocytes express both ferritin and IBA1. The inset in (d) shows matching single-color images in further support of the colocalization of IBA1 (Alexa Fluor 488 green) and ferritin (Cy3 red). Bars (all): 20 μm
Fig 2: Immunohistochemistry and immunofluorescence of the gap junction protein connexin 43 and the satellite cell marker S100 in DRG of normal controls and FA. a-d normal control; e-h FA; a and e positive contrast immunohistochemistry of connexin 43; b and f Alexa 488 immunofluorescence of S100; c and g Cy3 fluorescence of connexin 43; d and h, merged images of b and c, and f and g, respectively. In the normal DRG, connexin 43 shows punctate and coalescing reaction product (a; and inset [arrow]). In FA, a great abundance of connexin 43 reaction product is present in multiple layers around small neurons (e). Hyperplastic satellite cells bridge gaps between neurons. The confocal images of a normal DRG show sparse gap junctions by their punctiform connexin 43 immunofluorescence (c). They are closely related to S100-positive satellite cells (d). In FA, the number of connexin 43-reactive puncta is greatly increased (g and h). Bars: (a) and (e), 20 µm; insets, 10 µm; (b-d) and (f-h), 10 µm. N, neuron
Fig 3: Immunohistochemistry of glutamine synthetase, S100, and laminin in DRG of normal controls and FA. a-c normal controls; d-f FA; a and d glutamine synthetase; b and e S100; c and f laminin. In normal DRG, reaction product of glutamine synthetase (a) and S100 (b) reveal well organized layers of adjacent and overlapping satellite cells around each neuron. Normal nerve cells are surrounded by delicate layers of laminin (c). In FA, the remaining neurons are smaller, and satellite cell layers are thicker (d and e, insets) and disrupted (d, inset). Laminin reaction product shows multiple layers surrounding neurons, which is consistent with proliferation of satellite cell processes (f, inset). The arrows in (d) and (e) point to residual nodules that are reactive with antibodies to glutamine synthetase (d) and S100 (e), respectively. Bars: (a-f), 100 μm; insets, 20 μm
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