Journal: The Journal of Experimental Medicine

Article Title: Loss of direct adrenergic innervation after peripheral nerve injury causes lymph node expansion through IFN-γ

doi: 10.1084/jem.20202377

Figure Lengend Snippet: Sympathetic nerves curb LN responses. (a) Identification of sympathetic (red, TH) and nociceptive sensory (green, CGRP) nerves together with neurofilament (blue, NF) in the sciatic nerve. Scale bar, 200 µm. (b) Innervation of nociceptive sensory (left) and sympathetic (right) nerves to the popLN (circled), costained with CD31. Scale bar, 200 µm. (c) Innervation of sympathetic nerves to the popLN in untreated sham conditions, after systemic 6-OHDA treatment, local phenol treatment, or femoral (Fx), sciatic (Sx) or femoral and sciatic dual denervation (FSx) surgeries. Scale bars, 50 µm. (d) Cellularity of popLNs 1 wk after unilateral sciatic denervation in combination with substance P (SP), CGRP, or combined treatment (S+C); n = 5 or 6 mice from two independent experiments; two-way ANOVA; Šídák’s post-test. (e) Cellularity of the popLN 1 wk after unilateral sciatic denervation in combination with PBS or clenbuterol treatment; n = 4 mice; data are representative of two independent experiments; two-way ANOVA; Šídák’s post-test. (f) Cellularity of the popLN in Adrb2 −/− mice 1 wk after unilateral sciatic denervation in combination with PBS or clenbuterol treatment; n = 4 mice; data are representative of two independent experiments; two-way ANOVA; Šídák’s post-test. Adrb2 −/− mice were 8–12-wk-old males and bred in our animal facility. All other mice in this figure were 8–12-wk-old WT males purchased from Charles River or Janvier Labs. All data are presented as mean ± SEM; *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Clen, clenbuterol; Veh, vehicle.

Article Snippet: Samples were fixed in 4% paraformaldehyde (PFA) for 30 min. After fixation, samples were immersed in the blocking cocktail containing 20% goat serum (31872; Invitrogen), 0.5% Triton X-100 (M143; Amresco) and 20% (vol/vol) streptavidin solution (SP-2002; Vector Laboratories) in PBS for 2 h. For primary antibody staining, anti-TH (1:1,000) and anti-CD31–Alexa 647 (1:100) antibodies, 2% goat serum, and 20% (vol/vol) biotin solution (SP-2002; Vector Laboratories) in PBS were applied on samples for overnight staining.

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