| Summary: | Supplementary figure 2 Effect of extended crosslinking on antibody penetration in single cultured cells (MCF10A, non-transformed immortalized breast epithelium). Cells were immunostained for detection of 53BP1 nuclear protein. A minor fraction of the protein is also present in the cytoplasm. Supplementary Material and Methods for Supplementary figure 2: Cell culture, Immunostaining and Microscopy MCF10A non transformed immortalized breast epithelium cells were grown in DMEM 1 Ham’s F12 Medium (1:1) containing 5% FBS, 2 mM glutamine, 50 ng/ml penicillin/streptomycin (all from Lonza, Switzerland), cholera toxin (Sigma-Aldrich, MO), 10 µg/ml insulin (Roche, Switzer- land), 100 µg/ml hydrocortisone (Sigma-Aldrich), and 20 ng/ ml EGF (PeproTech, NJ) at 378Cin 5%CO2. Cells were grown on glass coverslips coated with 0.5% gelatin (wt/vol) in PBS. Exponentially growing cells were fixed in 4% paraformaldehyde (wt/vol) at room temperature (RT) for 10 min (standard conditions), 4h and for an overnight (18 h) to detect effects of crosslinking duration on immune-stainability. Fixed MCF10A cells were washed and permeabilized for 10 min in a permeabilization buffer containing 0.1% Triton X- 100 (vol/vol) in PBS. Coverslips were then immersed for 30 min in a blocking solution, 5% BSA (wt/vol) in PBS, then incubated for 1 h at RT with rabbit anti 53BP1 primary antibody (ab36823, Abcam) diluted 1 to 100 in blocking solution. After washes cells were incubated with Alexa 647-conjugated donkey anti rabbit secondary antibody (Jackson ImmunoResearch). Washed coverslips were then stained with DAPI (ThermoFisher) and mounted in Mowiol-containing mounting media for widefield fluorescence microscopy analysis. Images were acquired with a Nikon Ti2 inverted microscope equipped with an Orca Flash 3 camera (Hamamatsu) employing a 20x 0.75 NA objective. Confocal stacks were acquired to eliminate out-of-focus signals employing an A1R confocal scanhead with a 60x oil immersion 1.4 NA objective. Acquired images have been processed by the open-source ImageJ software (W. Rasband, NIH, http://imagej.nih.gov/ij). Widefield images were corrected by background subtraction using the Rolling-Ball algorithm included in the program. Intensity has been measured by thresholding whole cells and reporting the mean-pixel value. Nucleus vs Cytoplasm Ratio has been calculated measuring respectively the mean pixel values on a nuclear and cytoplasmic Region Of Interest for each cell. Maximum Z projection have been produced in ImageJ to provide a representation of the whole cell volume.
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