Low melting temperature agarose

Additionally, viral titers were evaluated by standard plaque assay. Briefly, Sf9 cells were seeded in 6-well plates at a density of 1x10⁶ cells/well. Supernatants from co-infections were 10-fold serially diluted in duplicates and 500 μL of each virus dilution was added to the cells. Plates were incubated for 1 h at 27°C, before the viral inoculum was removed and wells were overlaid with 2 mL of cell culture medium supplemented with 1% low melting temperature agarose (Sigma-Aldrich), 10% FBS (Gibco) and 1 x Antibiotic-antimycotic mix (Gibco). After incubation for 6 days at 27°C in a humid environment, plaque assays were stained with 1 mg/mL MTT (Sigma-Aldrich), plaques were counted, and the titer determined as PFU/mL. One-way ANOVA was applied to evaluate statistical significance.

Soft-agar assay was performed by seeding 5 × 10³ breast cancer cells stably expressing the indicated shRNA or cDNA constructs onto 0.4% low-melting-temperature agarose (Sigma-Aldrich) layered on top of 0.8% agarose. After 3–4 weeks of incubation, colonies were stained with 0.005% crystal violet and imaged using a microscope. Colony sizes were measured using ImageJ software (https://imagej.nih.gov/ij/) and plotted. Statistical analyses were performed using Student's t-test in GraphPad Prism 7 software (GraphPad, San Diego, CA, USA).

Mouse LNs were blocked and permeabilized overnight in PBS with 5% mouse serum, 5% rat serum, and 0.2% Triton X-100, followed by staining for 3 days at 37 °C with 1:100 dilutions of BV421-labeled rat anti-mouse CD35 clone 8C12 (BD Biosciences) and Alexa Fluor 488-labeled mouse anti-Ki67 clone B56 (BD Biosciences) in the same buffer as the blocking/permeabilization step. Stained LNs were then washed for 3 days at room temperature with PBS containing 0.2% Tween-20 and then the fixation protocol was repeated prior to whole-organ clarification.
Selected RM LNs were embedded in 3% low melting temperature agarose (Sigma-Aldrich) and then sliced into 100- or 350-µm-thick sections using a vibratome. The slices were blocked and permeabilized overnight in PBS with 5% mouse serum, 5% rat serum, and 0.2% Triton X-100, followed by staining for 3 days at 37 °C with 1:100 dilutions of BV421-labeled mouse anti-human CD35 clone E11 (BD Biosciences) and Alexa Fluor 488-labeled mouse anti-Ki67 clone B56 (BD Biosciences) in the same buffer as the blocking/permeabilization step. Stained slices were then washed for 3 days at room temperature with PBS containing 0.2% Tween-20 and then mounted onto glass slides with Prolong Glass antifade mountant (Thermo Fisher Scientific).

For live imaging in the light-sheet microscope, 48 hpf zebrafish embryos were anesthetized using 0.016% tricaine (Sigma) and then were embedded in 1.3% low-melting-temperature agarose (Sigma; prepared in filtered fish facility water) inside a glass capillary [1.5/2.0-mm inner/outer diameter, 20-mm length (Zeiss)]. The embryos were centered in the capillary and oriented. After gel formation, the section of the agarose cylinder containing the tail of the embryo was extruded from the capillary by inserting wax into the capillary on the side opposite to the fish. The sample chamber of the light-sheet microscope was filled with filtered fish facility water, and the capillary was inserted for imaging. Specimens were maintained at 32°C throughout the imaging period. Fluorescent image acquisition was performed using a Zeiss Lightsheet Z.1. Z-stacks were processed for maximum intensity projections with the Zeiss ZEN Software. For timelapse (4D) images, zstacks were taken every 5–15 minutes for a total time of up to 24 hours with a step number between 50 and 200 and step size of 0.3–2.0 μm. Images were adjusted for brightness and contrast using the Zeiss ZEN Software. Confirmation of injected cell migration from inside of the lumen to surrounding tissue was done using the Zeiss ZEN Software 3D retendering capability.