Immunostaining Protocol for Thoracic Mammary Glands

Thoracic mammary glands were fixed for 24 hours in 4% paraformaldehyde and embedded in paraffin wax. Paraffin sections of 5 µm were prepared and subjected to 1 mM disodium-EDTA antigen retrieval as described previously [28] (link). Primary antibodies used for immunofluorescence are the following: Cytokeratin 8 (Developmental Studies Hybridoma Bank TROMA-I, rat, 1∶100), Estrogen receptor (Novocastra NCL-ER-6F11, mouse, 1∶100), E-Cadherin (BD Biosciences 610181, mouse, 1∶250), Progesterone receptor (Abnova MAB9785, rabbit, 1∶400), Smooth muscle actin (Sigma A2547, mouse, 1∶1000), Tbx3 (Invitrogen 424800, rabbit, 1∶100), turboGFP (Pierce Antibodies PA522688, rabbit, 1∶400), turboGFP (OriGene TA150041, mouse 1∶250). Secondary antibodies used at 1∶400 dilution are from Invitrogen: 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 (Biotium 20137-1) was used at 1∶400 dilution. Images were acquired on a Zeiss LSM-710 confocal microscope with a pinhole aperture of 1 Airy unit.

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Kunasegaran K., Ho V., Chang T.H., De Silva D., Bakker M.L., Christoffels V.M, & Pietersen A.M. (2014). Transcriptional Repressor Tbx3 Is Required for the Hormone-Sensing Cell Lineage in Mammary Epithelium. PLoS ONE, 9(10), e110191.

Publication 2014

Cytokeratin 8 E-Cadherin Smooth muscle actin turboGFP Alexa488-coupled goat anti-rabbit CF633nm-coupled donkey anti-rat LSM-710 confocal microscope

Actin Airy Antibodies Antigen Confocal microscope Cytokeratin 8 Dilution Disodium edta Donkey E cadherin Estrogen receptor Goat Hybridoma Immunofluorescence Mammary glands Mouse Paraffin Paraformaldehyde Progesterone receptor Rabbit Smooth muscle

Corresponding Organization : National University of Singapore

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