Superfrost plus

Mice were deeply anesthetized and transcardially perfused with 4% PFA in PBS. Plantar skin was collected and post-fixed for 1hr at room temperature. Following fixation, tissue was immersed in 30% sucrose/PBS (overnight) and then mounted in OCT. Serial sagittal sections (10μm thick) were cut with a microtome and immediately collected onto slides (Superfrost Plus–VWR) coated with 2% gelatine. Slides were rinsed in water, dipped in 0.5% toluidine blue solution (pH 4) for 2 minutes, washed with water and mounted with DPX. Mosaics of single plane images were captured on using Axiovision LE Software, Axioskop microscope (Zeiss, Germany) with a 20× 1.3 NA objective. Images (at least 4 mosaics per animal) were analysed by counting positive cells and normalised by the length of the skin sample.

Cell-seeded composite scaffolds were fixed in 4% paraformaldehyde
for 45 minutes at room temperature followed by washing 3x in PBS for 5
minutes each. The scaffolds were soaked overnight in 30% w/v sucrose
solution, then soaked overnight in a 30% v/v solution of Optimal Cutting
Temperature (OCT; Tissue-Tek, Sakura Finetek, Torrance, CA) medium in 30%
w/v sucrose. Finally, the scaffolds were soaked overnight in pure OCT,
mounted in Cryo-Gel (Electron Microscopy Sciences, Hatfield, PA) in 10 mm
× 10 mm plastic cryomolds (Tissue-Tek), and frozen in liquid
nitrogen. Scaffolds were sectioned at −20° C in a Leica
CM1850UV cryostat (Leica Microsystems, Buffalo Grove, IL) at either 30
μm thick (for histological staining) and 60 μm thick (for
immunofluorescent / confocal imaging). Sections were mounted on
electrostatically charged glass slides (Superfrost Plus, VWR, Radnor, PA)
and heated on a slide warmer at 50° C for 30 minutes. Cryo-Gel was
dissolved from the sections by soaking slides in diH₂0 for 20
minutes.

A known number of stained cancer cells (1000 cells, measured with the Cellometer System (Nexcelom, Bioscience, USA) was spiked into vials filled with 500 μL RPMI. Subsequently, different quantities (0.5, 5.0, 25 or 50 μL) of anti-EpCAM or anti-CD45 PMPs were added to these samples. Next, these cell suspensions were admixed on a shaker at 4 °C for 30 min, while gently twisting and shaking to allow binding.
After 30 min, the prepared cell solution was placed in the input well, and the magnet was placed under this well. With slow advancement of the magnet under the channel toward the output well, bound cells of interest traversed the immiscible oil channel and entered the output well. The syringe was then used to extract the washing solution by passing it through the filter. Once these processes were complete, the filter was removed and handled over on a glass slide (VWR Superfrost Plus, USA), and Diamond anti-fade mounting media containing DAPI (Life Technologies, Grand Island, NY) was used to coverslip the slides.