Alexa fluor 594 donkey anti mouse a21203

The following antibodies were used: anti-HERC2 monoclonal (BD Biosciences); anti-HERC2 polyclonal [32 (link)]; anti-p21 (C-19); anti-p62 (SQSTM1 (D-3): sc-28359); anti β-actin (Santa Cruz Biotechnology, Inc.); anti-calbindin D-28k polyclonal (Cb-38a, Swant); anti-calbindin D-28k monoclonal (Cb-955, Sigma); anti-p53 Ab-5 (DO-7) (Neo Markers); anti-USP33 (Proteintech); anti-Ran [62 (link)]; anti α-tubulin (Ab-1, Calbiochem); Alexa Fluor^® 488 donkey-anti-rabbit (A21207), and Alexa Fluor^® 594 donkey-anti-mouse (A21203) (Invitrogen); horseradish peroxidase-conjugated secondary antibodies (Invitrogen); biotin-conjugated secondary antibodies (Vector); and the Avidine-Streptavidine Elite Kit (Vector).

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).