Upper panels: composite image collected with ×1.6 objective for ImageIT-Green (green excited at 514 nm) and vasculature (blue determined from transmission images). As a positive control, LNs were made anoxic by bubbling PBS with N 2 and including the O 2-scavenger dithionite (1 mM), followed by cessation of circulation by cervical dislocation. k Intravital imaging for hypoxic regions using 12.5 nmoles of ImageIT-Green hypoxic probe injected into B6 mice via the footpad in a 50 µl volume. Mean ± SEM pHe measured in B6 ( n = 6) and BALB/c ( n = 5) mice. pHe maps in inguinal LN region-of-interest are overlaid on anatomical T 2-weighted images.
j MRI-CEST pH imaging of control (B6) mice injected via i.v. i Summary data for each LN compartment from 4, 3, 4, 3 LNs, respectively. Note that compared to footpad injections, tail-vein injections detect an additional compartment corresponding to blood vessels. Plots shows the pH-distribution in each of the LN compartments, averaged for all LNs. h Statistical distribution of pHe data analyzed by Gaussian mixed models to separate pixels into clusters, representing compartments. Measurements on control mice, or mice treated with LPS ( n = 4). Mice were injected with 70 kDa dextran-conjugated cSNARF1 into the tail-vein (20 mg/ml in 100 µl). g Intravital imaging of pH-sensitive cSNARF1 fluorescence in inguinal LN. Significance tested by one-way ANOVA with multiple comparisons ( N = 5, 10, 4, 4, 8, 11) two sided at 5% significance.
f Summary data for mean pHLIP fluorescence within LN boundary. e Experiment performed on athymic nude mouse, with same imaging settings, showing absence of pHLIP signal ( n = 8). or d 200 µl of 5.2 mg/kg (5-N,N-dimethyl)amiloride (DMA) and 3.9 mg/kg acetazolamide (ATZ) 24 h prior to imaging ( n = 3). Experiment repeated on B6 mouse injected via the intraperitoneal cavity with c 200 µl of 12.5 mg/kg omeprazole (OME) and 1 mg/kg of bafilomycin (BAF) ( n = 4). Right: montage of pHLIP fluorescence collected in overlapping fields of view with ×10 objective, summed across the depth of the LN ( n = 10 mice). Left: composite image collected with ×1.6 objective for pHLIP-Cy5.5 (red excited at 633 nm), autofluorescence (green excited at 514 nm) and vasculature (blue determined from transmission images). b B6 mouse injected with pHLIP (40 µM in 60 µl) into footpad, followed by intravital imaging of inguinal LN 24 h later in window chamber. Histological section of inguinal LN showing T-cell marker CD3 in paracortical zone ( N = 3).
A Cartoon of lymph node (LN) showing zones occupied by T and B cells, blood vessels (B.V.) and the medulla (Me).
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