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anti-L-selectin monoclonal antibody Mel-14 (100 mg per mouse). At various time points during the following 48 h, mice were anaesthetized by an initial intraperitoneal injection of
 

Summary: anti-L-selectin monoclonal antibody Mel-14 (100 mg per mouse). At various time points
during the following 48 h, mice were anaesthetized by an initial intraperitoneal injection of
ketamine (50 mg kg21
) and xylazine (10 mg kg21
). The right popliteal LN was prepared
microsurgically for intravital microscopy and positioned on a custom-built microscope
stage. Care was taken to spare blood vessels and afferent lymph vessels. The prepared LN
was submerged in normal saline and covered with a glass coverslip. A thermocouple was
placed next to the LN to monitor local temperature, which was maintained at 36 8C. In
some experiments the LN microcirculation was revealed by intravenous injection of 2%
tetramethylrhodamine b-isothiocyanate­dextran or fluorescein isothiocyanate­dextran
(500 or 2,000 kDa; Molecular Probes). Two-photon imaging was performed with an
Olympus BX50WI fluorescence microscope equipped with a 20£, 0.95 numerical aperture
objective (Olympus) and a Bio-Rad Radiance 2000MP Confocal/Multiphoton microscopy
system, controlled by Lasersharp software (Bio-Rad). For two-photon excitation and
second harmonic generation, a Tsunami Ti:sapphire laser with a 10-W MilleniaXs pump
laser (Spectra-Physics) was tuned to 800 nm.
For four-dimensional analysis of cell migration, stacks of six squared x­y sections with
6 mm z spacing were acquired every 15 s with electronic zooming up to 4£ to provide
image volumes 30 mm in depth and 154­618 mm in width. Emitted light and second

  

Source: Amasino, Richard M. - Department of Biochemistry, University of Wisconsin at Madison

 

Collections: Biology and Medicine