To calculate the mitotic index of basal and luminal cells, paraffin sections of mammary glands from BrdU-injected mice were counterstained with lineage-specific markers (K5 and K8), together with a nuclear stain (to score the total cell number of each cell type).
Secondary antibodies and reagents were: AlexaFluor 488 goat anti-mouse, AlexaFluor 546 goat anti-rat, AlexaFluor 488 goat anti-rabbit, AlexaFluor 647 goat anti-rabbit, Pacific Blue conjugated goat anti-rabbit (all from Invitrogen, Carlsbad, CA). Primary antibodies are: antiKi67 (BD Biosciences, Franklin Lakes, NJ), Keratin 5 Clone AF 138 (Covance, Emeryville, CA), Keratin 8 (The University of Iowa ?Developmental Studies Hybridoma Bank, Iowa City, Iowa), cH2AX (Millipore, Billerica, MN), BrdU (Roche, Indianapolis, IN), phospho-Ser15 p53 (Cell Signaling Technology, Beverly, MA). General MedChemExpress 6 Methods for immunohistochemical analysis of paraffin-embedded mammary samples include antigen retrieval as described. Secondary antibodies were peroxidase-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and horseradish peroxidase conjugated anti-rabbit IgG (InVitrogen).Whole Mount Staining and ImmunohistochemistryMammary glands were fixed and processed to visualize ductal trees as described. Antibodies used as probes were: anti-phospho-Lrp6 (detecting both phospho-Lrp6 and phospho-Lrp5 Cell Signaling Technology), anti-vinculin (Millipore), and anti-b-actin (clone AC-15, Sigma-Aldrich). The resulting tissue and cell lysates were cleared for 20 min at 10 000 g at 4uC and the supernatant was analyzed by Western blotting. The resulting powder was mixed with high salt buffer containing protease and phosphatase inhibitors and homogenized for ten seconds, twice, at 4uC with a Polytron (Kinematica, AG, Switzerland). For tissues, mammary glands were snap frozen in liquid nitrogen and ground with a mortar and pestle. `Cell and Tissue Preparation for Western Blot AnalysisCells were rinsed with PBS and scraped into a high salt lysate buffer (25 mM HEPES pH 7.4, 300 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, and 0.5 Triton X100, with Halt protease and phosphatase inhibitor cocktails, Pierce, Rockford, IL). S1A and B, and were generated according to. The gating profiles and trees are shown in Fig.
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Nces) equipped with FlowJo software (Tree Star Inc.
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