Fv10i flouview ver 3

Multiplexed IF staining was performed using Opal kit as per the manufacturer’s protocol and as described previously (23 (link)). Briefly, 5µm frozen sections were exposed to epitope retrieval, blocked, and incubated with primary antibody (1 h at room temperature), and then horseradish peroxidase and the secondary antibody were introduced. After the signal amplification with fluorophores, the slides were counterstained with DAPI, and mounted using ProLong™ diamond antifade mountant. Then the coverslipped sections were examined by confocal microscopy and imaged on Olympus Fluoview Fv10i confocal microscope. Analysis was performed using fv10i flouview Ver.3.0 software (Olympus Europa Holding GmbH, Hamburg, Germany). For IF microscopy, stained tissue was imaged by Olympus microscope and analyzed using NIS-Elements Ar software (Nikon Inc., NY, USA).

Mammary gland cryosections were processed as described above for immunohistochemistry and evaluated by confocal microscopy. HMEpCs were plated in 8-well chamber slides (Nunc, Thermo Fisher Scientific) at 1 × 10⁴ cells per well and were allowed to adhere overnight. Cells were washed with PBS, fixed for 15 min with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 for 10 min. For both tissues and cells, nonspecific sites were blocked for 1 h at room with 3% BSA, washed in PBS-T and incubated at 4 °C overnight with primary antibodies: Notch 1 (antibody clone EP1238Y, ab52627), EEA1 (ab70521), TGF-β1 (ab66043), pSmad2 (ab188334) and Ki67 (ab15580) were from Abcam, Cambridge, MA, USA; cytokeratin 18 (Fitzgerald, North Acton, MA, USA; 70R-30585) and cytokeratin 14 (Thermo Fisher Scientific; MA5-11599). After primary antibody incubation, slides were washed three times with PBS and incubated with secondary antibodies: Alexa Fluor 488 goat anti-rabbit (A-11008) and Alexa Fluor 594 donkey anti-mouse (A-21203) (Invitrogen, Thermo Fisher Scientific) for 1 h at room temperature. The stained slides were imaged on an Olympus Fluoview Fv10i confocal microscope (Olympus, Waltham, MA, USA). Analysis was performed using Fv10i Flouview Ver.3.0 software (Olympus).