Lavision ultramicroscope

Mice were sacrificed and lymph nodes were dissected and fixed in 4% PFA. After a wash in PBS, samples were permeabilized in 0.5% Triton-X 100 in PBS. After a wash in PBS, samples were blocked in blocking solution (1% BSA, 0.1% Tween-20 in PBS) and subsequently incubated with primary antibodies diluted in blocking solution. The antibodies used were rabbit anti-RFP (1:100, Rockland) and goat anti-LYVE-1 (1:100, R&D Systems). Samples were washed in PBS 0.1% Tween-20 (PBS-T) and incubated with AlexaFluor 488 and 647-conjugated secondary antibodies (1:200, Invitrogen) in blocking solution. After a wash in PBS-T, samples were embedded in 1% ultrapure low melting point agarose, dehydrated with methanol and cleared with BABB (benzyl alcohol/ benzyl benzoate 1:2) [20 (link)]. Briefly, samples were dehydrated in a series of 50%, 70%, 95% (at least 1 hour each) and 100% methanol (overnight) and cleared for 1 hour in 50% BABB in methanol and finally in BABB for at least 8 hours. Samples were stored in BABB at 4°C in the dark until image acquisition. Images were acquired using a LaVision Ultramicroscope (LaVision BioTec, Bielefeld). Stacks were captured using 2 μm step size and 2.5x magnification. Maximum projection of a 260 μm z-stack was obtained using the ImageJ software.

Immunostained and optically cleared liver samples were imaged on a LaVision Ultramicroscope (LaVision BioTec, Bielefeld) with a step size of 2 µm. 3D reconstruction was performed on high end commodity hardware using the volume rendering software package Voreen (voreen.uni-muenster.de) (Meyer-Spradow et al., 2009 (link); Hägerling et al., 2017 (link))