Uplfln10x2

Sections were viewed by immunofluorescence microscope (BX61, Olympus, Tokyo, Japan) and captured using a cooled digital camera (DP71, Olympus, Tokyo, Japan). The objectives used were: UPLFLN4X, UPLFLN10X2, UPLFLN40X, and UPLFLN100XO2 (all Olympus, Tokyo, Japan). In order to quantify Cx immuno-expression, visual fields captured at an objective magnification of 40× and constant exposure times were analyzed. Green granular deposits were interpreted as positive Cx37, Cx43, and Cx45 immuno-expression. Quantification of immunoreactivity was performed by using ImageJ (National Institutes of Health, Bethesda, MD, USA). Figures were prepared for analysis by using subtraction of the median filter 2 px and thresholded by using the color triangle threshold algorithm. Section percentage area covered by Cx immunofluorescence was measured in Adobe Photoshop, by manual outlining of the areas of interest. For the purpose of presentation, background subtraction and contrasting of microphotographs were conducted.

Temperature sensors (liquid crystal thermometers; Type C 30−60 °C with 5 °C intervals from ThermometerSite) were placed directly under the hydrogel lid (immersed in F-actin stabilization lysis buffer). The temperature was monitored while applying 30 V cm⁻¹ across the electrodes of the electrophoresis chamber without interfacing with the custom heater.
Fluorescence imaging of cells in microwells, lysis, and PAGE: Imaging was performed via time-lapse epi-fluorescence microscopy on an Olympus IX50 and IX51 inverted epifluorescence microscope (and thus the custom heater was not used as it would block the illumination path through the PAGE chamber). The microscope was controlled using Metamorph software (Molecular Devices) and images were recorded with a CCD camera (Photometrics Coolsnap HQ2). The imaging setup included a motorized stage (ASI), a mercury arc lamp (X-cite, Lumen Dynamics), and a XF100-3 filter (Omega Optical) and 41017 (Chroma) for GFP and an XF111-2 filter for RFP (Omega Optical). Imaging was performed with a 10× magnification objective (Olympus UPlanFLN, NA 0.45 or UPLFLN10X2, NA 0.3) and 900 ms exposures with 1 s intervals with U2OS RFP-Lifeact, and 2 s exposure with 2 s intervals with MDA-MB-231 GFP-actin (1× pixel binning). Exposure times were lowered for lysis imaging to 600 ms.