Dylight 594 labeled lectin

After euthanasia, LNs from mice were surgically dissected and transferred to a Chamlide AC-B25 imaging chamber (Live Cell Instruments) with a customized coverslip platform to allow flow beneath the LN. The LN was stabilized with a tissue slice harp (Warner Instruments) and superfused with oxygenated Dulbecco’s Modified Eagle’s Medium (Gibco, 21063-045) and maintained at 37°C. For experiments in which blood vessels were imaged in conjunction with T cells or DCs, with 70 μg DyLight 594-labeled lectin (from L. esculentum, Vector Laboratories) was intravenously administered by tail vein injection 5 min before euthanasia.

Selected phage clones and insertless phage were i.v. injected via tail vein (2 × 10¹⁰ phage/animal in 300 μl PBS). 10 minutes before perfusion and subsequent fixation 100 ul DyLight594 labeled lectin (Vector Laboratories Inc., DL-1177) was i.v. injected into the same animals. 60 minutes after phage administration rats were perfused through the heart with 50 ml PBS followed by 50 ml 4% PFA/PBS. Brain samples were additionally fixed by 4% PFA/PBS overnight and immersed in 30% sucrose at 4 °C overnight. Samples were frozen quickly in O.C.T. compound. Frozen samples were subject to immunohistochemistry, performed on 30 um freezing sections blocked with 1% BSA for 2 hours at room temperature and incubated with a polyclonal FITC labeled antibody against the T7 phage (Novus NB 600-376A) overnight at 4 °C. Finally, the sections were then washed 3 times with PBS and observed with a laser confocal microscope (Leica TCS SP5).

The lung vasculature of LPS-treated mice was labeled intravenously (i.v.) with 70 µg DyLight594-labeled lectin (from Lycopersicon esculentum, Vector Laboratories). After euthanasia, the lungs were surgically dissected and inflated intratracheally with low gelling temperature agarose (2% dissolved in PBS, Sigma)^{29 (link)}. After transfer into a customized imaging chamber (for imaging with the Bio-Rad microscope) or a Chamlide AC-B25 imaging chamber (Live Cell Instruments; for the ZEISS microscope), mechanically stabilized lungs were superfused with oxygenated DMEM (Dulbecco's Modified Eagle's medium) (Gibco, 21063-045; 5% serum, Atlanta Biologicals; 100 units ml⁻¹ penicillin and 100 µg ml⁻¹ streptomycin, Gibco) and maintained continuously at 37 °C.