Fixed #3 mammary glands were processed and embedded in paraffin wax. Paraffin sections of 5 μm were prepared and subjected to 1 mM disodium-ethylenediaminetetraacetic acid (EDTA) antigen retrieval as described previously
[21 (link)]. Primary antibodies used for immunofluorescence are the following: cytokeratin-8 (CK8) (TROMA-I, rat, 1:100; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), estrogen receptor (NCL-ER-6 F11, mouse, 1:100; Novocastra, which is part of Leica, Wetzlar, Germany), GFP (600-401-215, rabbit, 1:100; Rockland Immunochemicals Inc., Gilbertsville, PA, USA), GFP (600-141-215, goat coupled to Dylight-488, 1:300; Rockland Immunochemicals Inc.), progesterone receptor (MAB9785, rabbit, 1:400; Abnova, Taipei, Taiwan), and smooth muscle actin (SMA) (A2547, mouse, 1:1,000; Sigma-Aldrich, St. Louis, MO, USA). Secondary antibodies used at 1:400 dilution are from Invitrogen Corporation: Alexa488-coupled goat anti-mouse (A11029), Alexa488-coupled goat anti-rabbit (A11034), Alexa568-coupled goat anti-mouse (A11031), and Alexa568-coupled goat anti-rabbit (A11036). Additionally, CF633nm-coupled donkey anti-rat (20137–1; Biotium, Hayward, CA, USA) was used at 1:400 dilution.
[21 (link)]. Primary antibodies used for immunofluorescence are the following: cytokeratin-8 (CK8) (TROMA-I, rat, 1:100; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), estrogen receptor (NCL-ER-6 F11, mouse, 1:100; Novocastra, which is part of Leica, Wetzlar, Germany), GFP (600-401-215, rabbit, 1:100; Rockland Immunochemicals Inc., Gilbertsville, PA, USA), GFP (600-141-215, goat coupled to Dylight-488, 1:300; Rockland Immunochemicals Inc.), progesterone receptor (MAB9785, rabbit, 1:400; Abnova, Taipei, Taiwan), and smooth muscle actin (SMA) (A2547, mouse, 1:1,000; Sigma-Aldrich, St. Louis, MO, USA). Secondary antibodies used at 1:400 dilution are from Invitrogen Corporation: Alexa488-coupled goat anti-mouse (A11029), Alexa488-coupled goat anti-rabbit (A11034), Alexa568-coupled goat anti-mouse (A11031), and Alexa568-coupled goat anti-rabbit (A11036). Additionally, CF633nm-coupled donkey anti-rat (20137–1; Biotium, Hayward, CA, USA) was used at 1:400 dilution.
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