Visualizing Lymph Node Responses to Vaccines

Mouse LNs were isolated 72 h post-intradermal injection of model vaccines. LNs were fixed with 4% paraformaldehyde (Thermo Scientific, Rockford, IL) and incubated in 30% sucrose solution (Merck, Germany) overnight. Ten-μm thickness of frozen sections were cut by a cryostat (Leica, Germany). Images were acquired on a 4-channel NIR fluorescence microscope (TE2000U, Nikon, Japan) and CCD camera (C4742–80-12AG, Hamamatsu Photonics, Japan). All the images were binarized by means of iterative selection method to define areas positive for fluorescence signals. To analyze the spatial relationship between fluorescent cells and their locations in the LN, areas within 15 μm of the edge of LNs were defined as subcapsular sinus (SCS) and the others were considered as cortex.^{[34 (link)]} The portion of areas positive for fluorescence was measured and calculated for each ROI using ImageJ.