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3 protocols using rabbit anti cdh1

1

Immunofluorescent Staining of ISH-Stained Tissues

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Immunofluorescent staining of ISH-stained tissues and paraffin sections was performed as described previously. Primary antibodies were diluted as follows: 1:200 rabbit anti-CDH1 (3195, Cell Signaling Technology, Beverly, MA), 1:250 mouse anti-CDH1 (610181, BD Transduction Laboratories, San Jose, CA), 1:100 rabbit anti-phosphoSMAD1/5/8 (9511, Cell Signaling Technology, Danvers, MA). Secondary antibodies were diluted as follows: 1:250 Dylight 549-conjugated goat anti-rabbit IgG (111-507-003, Jackson ImmunoResearch, West Grove, PA), and 1:250 Dylight 488-conjugated goat anti-mouse IgG (115-487-003, Jackson ImmunoResearch). Immunofluorescently labeled tissues were counterstained with 4’,6-diamidino-2-phenylindole, dilactate (DAPI), and mounted in anti-fade medium (phosphate buffered saline containing 80% glycerol and 0.2% n-propyl gallate). Whole mount immunohistochemistry was performed as described previously. Primary antibody was diluted 1:750 rabbit anti-CDH1 and secondary antibody was diluted 1:500 biotin conjugated goat anti-rabbit IgG (BA-1000, Vector, Burlingame, CA).
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2

Immunofluorescent Staining of ISH-Stained Tissues

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Immunofluorescent staining of ISH-stained tissues and paraffin sections was performed as described previously (12 (link), 23 (link)). Primary antibodies were diluted as follows: 1:200 rabbit anti-CDH1 (3195, Cell Signaling Technology, Beverly, MA), 1:250 mouse anti-CDH1 (610181, BD Transduction Laboratories, San Jose, CA) 1:50 mouse anti-KRT14 (ms-115-p0, Thermo Fisher Scientific), 1:250 rabbit anti-AR (sc-816, Santa Cruz Biotechnology, Santa Cruz, CA), 1:200 rabbit anti-DNMT1 (5032, Cell Signaling Technology). Secondary antibodies were diluted as follows: 1:250 Dylight 549-conjugated goat anti-rabbit IgG (111-507-003, Jackson ImmunoResearch, West Grove, PA), 1:250 Dylight 488-conjugated goat anti-rabbit IgG (111-487-003, Jackson ImmunoResearch) and 1:250 Dylight 488-conjugated goat anti-mouse IgG (115-487-003, Jackson ImmunoResearch). Immunofluorescently labeled tissues were counterstained with 4’,6-diamidino-2-phenylindole, dilactate (DAPI), and mounted in antifade medium (phosphate buffered saline containing 80% glycerol and 0.2% n-propyl gallate). Whole mount immunohistochemistry was performed as described previously (23 (link)). Primary antibody was diluted 1:750 rabbit anti-CDH1 and secondary antibody was diluted 1:500 biotin conjugated goat anti-rabbit IgG (BA-1000, Vector, Burlingame, CA).
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3

Immunohistochemistry and in-situ Hybridization

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Tissues were fixed in 4% paraformaldehyde, embedded in paraffin wax, and sectioned at 5-μm intervals. Immunohistochemistry was performed using the following antibodies: mouse anti-phopho-histone3 (1:200; Cell Signaling Technology), goat anti-CC10 (1:20; Santa Cruz Biotechnology), rabbit anti-SP-C (1:500; Chemicon), rabbit anti-Nkx2.1 (1:50; Santa Cruz Biotechnology), mouse anti-Cdkn1a (1:100; Santa Cruz Biotechnology), rabbit anti-Sox9 (1:100; Santa Cruz Biotechnology), rabbit anti-Rbl2/p130 (1:50; Abcam), rabbit anti-Par3 (1:100; Upstate Biotechnology), rabbit anti-Sox2 (1:500; Seven Hills Bioreagents), and rabbit anti-Cdh1 (1:100; Cell Signaling). Slides were mounted with Vectashield mounting medium containing DAPI (Vector Laboratories). ISH was performed as described (Zhang et al. 2008 (link)). Probes were amplified from each lncRNA using the primers listed in Supplemental Table 8.
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