Journal: The Journal of Neuroscience
Article Title: The Meissner Corpuscle Revised: A Multiafferented Mechanoreceptor with Nociceptor Immunochemical Properties
Figure Lengend Snippet: Additional immunochemical characteristics of MC innervation as seen in conventional immunofluorescence digital images of MCs in 14-μm-thick sections cut perpendicular to the skin surface.ep, Epidermis; d, dermis. Each panel is an image double-labeled with two primary antibodies made in different species against the antigens indicated at the bottom. The fluorophore conjugated to the secondary antibodies is indicated by a red bar (Cy-3) or green bar (Cy-2) located beneath the antigen of the correspondingly labeled primary antibody. Based on morphology, location, and the total results of all double-label combinations used in the study (Figs. (Figs.3,3, ,4),4), the likely NF-positive Aαβ-fiber innervation is indicated byarrowheads; likely CGRP-positive C-fiber innervation is indicated by long straight arrows; and likely VR1-positive C-fiber innervation is indicated by curved arrows. Epidermal innervation is indicated by broad arrows in some panels. Red and green arrowheads and arrows indicate processes definitively labeled only by the antibody for the antigen listed over the red and green bars, respectively.Yellow arrowheads and arrows indicate definitively double-labeled innervation. Large arrowsand arrowheads indicate likely source axons;small arrows and arrowheads indicate presumptive terminals. For illustration purposes, the image intensities were digitally adjusted so that the maximum labeling intensity for each primary–secondary antibody combination was approximately the same. In many cases, contrast was increased, and low-end signals were subtracted to partially reduce the relatively high background labeling that occurs with some antibodies, the increased background caused artificially by digitally enhancing the overall image intensity, or both.A, Guinea pig anti-VR1 labeled C-fiber innervation (red curved arrows) is segregated from innervation labeled with rabbit anti-CGRP-IR. On the basis of other preparations (Fig. (Fig.33A–D), the segregated CGRP-positive innervation includes intermingled CGRP-positive endings of Aαβ-fibers (green arrowheads) and C-fibers (green long straight arrows). Some epidermal innervation expresses only VR1-IR (broad red arrow) or CGRP-IR (results not shown). B, Rabbit anti-P2X2 labeling (green arrows andarrowheads) is segregated from innervation labeled with guinea pig anti-VR1 (red curved arrows). As determined from other preparations (e.g., G), P2X2-IR is expressed on both the CGRP-positive C-fiber innervation (green arrows) and Aαβ-fiber innervation (green arrowheads). C, guinea pig anti-VR1 (red curved arrows) and rabbit anti-μOR (green straight arrows) label separate sets of innervation in the MC. On the basis of other label combinations (results not shown), the μOR-IR was definitive only on the CGRP-positive C-fiber innervation. A few spots of μOR-IR coincide with the VR1-positive innervation, but this was not consistent. Anti-μOR and anti-VR1 label mostly separate epidermal innervation (red and green broad arrows). D, Rabbit anti-δOR labels innervation (green arrows and arrowheads) in zones between the guinea pig anti-VR1-positive innervation (red curved arrows). Other label combinations (results not shown) revealed that the δOR-IR is expressed on the Aαβ-fiber innervation and on the CGRP-positive C-fiber innervation. A few spots of δOR-IR coincide with the VR1-positive innervation, but this was not consistent. E, Rabbit anti-NOCI labeling is coexpessed (yellow curved arrows) on guinea pig anti-VR1-positive innervation in the MCs. As determined from other preparations (e.g., K), other anti-NOCI labeling (red straight arrows) is likely on the CGRP-positive C-fiber innervation. Asterisks indicate VR1-negative zones where the Aαβ-fiber and CGRP-positive C-fibers terminate (Figs. (Figs.3,3, ,4).4). Some epidermal innervation is VR1-positive and NOCI-negative (green broad arrow).F, Rabbit anti-P2X1 labeling is highly punctate. Some is detectable on guinea pig VR1-positive innervation (yellow curved arrows) in the MCs. As determined in other double-label combinations (results not shown), P2X1-IR is also expressed on CGRP-positive C-fiber innervation (red straight arrows).Asterisks indicate VR1-negative zones where the Aαβ-fiber and CGRP-positive C-fibers terminate. VR1-IR and P2X1-IR can be expressed on separate epidermal innervation (redand green broad arrows). G, H, Rabbit anti-P2X2 and anti-P2X3 colabels with sheep CGRP-IR both on the CGRP-positive C-fiber innervation (yellow straight arrows) and on the Aαβ-fiber innervation (yellow arrowheads), as determined from other preparations. Both P2X2-IR and P2X3-IR are relatively fainter on the C-fiber innervation. Asterisks indicate CGRP-, P2X2-, and P2X3-negative zones where the VR1-positive innervation terminates (Fig. (Fig.4).4). Anti-P2X3 labeling is more readily detectable than anti-P2X2 on epidermal innervation, where it can be independent of CGRP expression (red and green broad arrows).I, Rabbit α2A-IR is coexpressed on sheep anti-CGRP-labeled C-fiber (yellow long arrows) as well as coexpressed more intensely on the Aαβ-fiber innervation (yellow arrowheads), as determined from other double-label combinations (results not shown). The α 2A-IR and CGRP-IR can be expressed on separate axons terminating in the epidermis (red and green broad arrows).J, Rabbit anti-α 2C labeling is coexpressed only on the CGRP-positive profiles that are the thicker endings of the Aαβ innervation (yellow arrowheads). CGRP-positive C-fiber innervation is present without α2C-IR in and around the MCs (green long arrows) and supplying the epidermis (green broad arrow). Asterisksindicate the CGRP and α2C-negative zones where the VR1-positive innervation would be located. K, Aαβ innervation labeled with mouse anti-NFp (green arrowheads) has little if any definitive rabbit anti-NOCI-IR. Anti-NOCI labels innervation (red curved arrows) between the Aαβ endings, which is where the VR1 innervation terminates (seeE), as well as innervation (red straight arrows) closely juxtaposed to the Aαβ endings, which is where the CGRP-positive C fibers terminate. L, Rabbit anti-VRL1-IR is only definitively colocalized with mouse anti-NFp labeling, indicative of the Aαβ-fiber innervation.
Article Snippet: Afterward, the sections were rinsed for 30 min in PBS and either processed for a second run of primary and secondary antibodies or coverslipped under 90% glycerol in PBS. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antigen Antibody (dilution) Source PGP9.5 Rabbit polyclonal (1:800–1500) UltraClone Ltd. (Wellow, UK) CGRP Sheep polyclonal (1:800–1500) Affiniti Research Products (Mamhead-Exeter, UK) Rabbit polyclonal (1:800–1500) Chemicon International (Temecula, CA) Neurofilament 200 kDa subunit Nonphosphorylated (NFn) Rabbit polyclonal (1:800–1500) Chemicon International Phosphorylated (NFp) Mouse monoclonal (RT97) (1:400–800) Chemicon International Glia-specific S100 protein Rabbit polyclonal (prediluted) Biogenesis (Poole, UK) MBP Mouse monoclonal (1:500–1000) Sternberger Monoclonals (Baltimore, MD) SP Guinea pig polyclonal (1:400–800) Research Diagnostics (Flanders, NJ) Substance P receptor (NK1) Rabbit polyclonal (1:1000–3000) Chemicon International Purinergic receptors P2X1 Rabbit polyclonal (1:1000–3000) Vulchanova et al., 1996 P2X2 Rabbit polyclonal (1:500–1500) Vulchanova et al., 1996 P2X3 Rabbit polyclonal (1:500–1500) Vulchanova et al., 1997 Opioid receptors κOR Rabbit polyclonal (1:1000–3000) Arvidsson et al., 1995c μOR Rabbit polyclonal (1:1000–3000) Arvidsson et al., 1995b δOR Rabbit polyclonal (1:1000–3000) Arvidsson et al., 1995a Adrenergic receptors Alpha 2A (α2A) Rabbit polyclonal (1:1000–3000) Stone et al., 1998 Alpha 2C (α2C) Rabbit polyclonal (1:1000–3000) Stone et al., 1998 Vanilloid receptors VR1 Guinea pig polyclonal (1:1000–3000) Guo et al., 1999 VR1 Rabbit polyclonal (1:1000–3000) Guo et al., 1999 VR1 Rabbit polyclonal (1:1500–5000) Caterina et al., 1997 VRL1 Rabbit polyclonal (1:1000–3000) Caterina et al., 1999 NOCI Rabbit polyclonal (1:1000–3000) Riedl et al., 1996 Open in a separate window List of antibodies used To control for nonspecific labeling, incubations were conducted without the primary antibodies and with primary antibodies preabsorbed with their specific blocking peptide.
Techniques: Immunofluorescence, Labeling, Expressing