Immunofluorescence Imaging of Mammary Tissue

Expression and distribution of various proteins were visualised by indirect immunofluorescence. Cells were fixed for 10 min in PBS/4% (w/v) paraformaldehyde and permeabilised for 7 min using PBS/0.2% (v/v) Triton X100. Nonspecific sites were blocked with PBS/10% goat serum (1 h, RT) prior to incubation with antibodies diluted in PBS/5% goat serum (1 h, RT, each). EdU was detected using the Click-iT EdU Alexa Fluor 647 imaging kit (Life Technologies), and nuclei were stained using 4 μg/ml Hoechst 33258 (Sigma) for 5 min at RT. Cells were washed in PBS before mounting in prolong gold antifade (Molecular Probes). Images were collected on a Nikon A1 confocal or a Leica TCS SP5 AOBS inverted confocal as previously described [15 (link)]. Nonbiased cell counts were performed by concealing the identity of each slide.
Immunostaining of mammary tissue was performed on paraffin-embedded tissue (5 μm) or cryosections (10 μm) as previously described [9 (link)]. Wheat germ agglutinin (WGA)-488, or -647 (Invitrogen, #W11261, #W32466), was used for staining the epithelium and imaged using confocal microscopy. Primary antibodies used for immunofluorescence are indicated in S2 Table. Secondary antibodies were conjugated to Cy2, Alexa-488, Rhodamine-RX, Cy5, Alexa-647 (Jackson Immunoresearch). Nonbiased image analysis was performed in 5 to 10 microscopy fields per mouse.