Dextran alexa fluor 647

Intracardial dextran injection was used for acute labeling of the vasculature in live tadpoles, as well as in assays for uptake of vascular-circulating molecules. For acute labeling of the vasculature, anesthetized tadpoles were arranged (ventral side up) under a dissecting microscope. Approximately 200-500 nl of solution containing 10,000 MW Alexa Fluor 647 dextrans (ThermoFisher Scientific, #D22914) at 25 mg/ml was pressure injected via glass micropipette into the heart. The entire volume of dextran solution is injected in a series of 5- to 10-nl pulses spaced over a minute. Tadpoles were imaged immediately after intracardial injection. For tadpoles imaged at multiple time points (with an interval of 24 h), intracardial dextran injection were performed daily. For dextran uptake experiments, 70,000 MW fluorescein dextrans (ThermoFisher Scientific, #D1822) were used (Hoffmann et al., 2011 (link)). After 5 h of recovery in 0.1× Steinberg’s solution, tadpoles were fixed and subsequently used for immunohistochemistry. For visualization of endocytic vesicles, injections were performed as described, except with 10,000 MW pHrodo red dextrans (ThermoFisher Scientific, #P10361). During the imaging interval, tadpoles anesthetized again and injected intracardially with 10,000 MW Alexa Fluor 647 dextrans (ThermoFisher Scientific, #D22914) to label the vasculature.

A431 cells, seeded onto glass-bottom dishes (Nunc LabTek 4-well chambered coverglass), were first labeled with 50 μg/ml Alexa Fluor 647-dextran (10,000 MW; Thermo Scientific) supplemented with 200 μM oleic acid/BSA in 5% LPDS to label late endosomal organelles and to induce LDs. The cells were then pulse-labeled for 2 h with 50 μg/ml BC LN-LDL in serum-free DMEM. During the last 30 min of LDL labeling, HCS LipidTox Red or HCS LipidTox Deep Red (1:1000; Thermo Scientific) was added to the medium to label LDs. The cells were then washed and chased in serum-free CO₂-independent medium (Gibco) for the indicated times and imaged by live-cell confocal microscopy. Imaging was performed on a Leica TCS SP8 X attached to a motorized DMi8 inverted microscope with ×63 HC PL APO CS2 water objective (1.20 NA). Experiments were performed at 37 °C in CO₂-independent medium (Gibco) supplemented with HCS LipidTox Red/Deep Red (1:1000) in a fully enclosed temperature-controlled environmental chamber. Data were acquired with Leica LAS X (Leica Microsystems). The fraction of BC residing in dextran-positive LEs and LipidTox-positive LDs was quantified from background-subtracted images with ImageJ by using Mander’s overlap coefficient as a measure of colocalization.