Alexa fluor 647 labeled goat anti rabbit igg

The HMC3 human microglial cell line (ATCC CRL-3304) and EMEM medium (ATCC 30–2003) were purchased from American Type Culture Collection (ATCC, Manassas, VA). BrdU (59–14-3) and mouse anti-BrdU (B2531) monoclonal antibody (mAb) were purchased from Sigma (St. Louise, MO). BAY 11–7082 was obtained from Cayman Chemical (Ann Arbor, MI, cat# 10010266). Human IL-6 DuoSet ELISA (DY206) kits were purchased from R&D Systems (Minneapolis, MN). Phospho-p53 (Ser 15) antibody (#9284), p53 mouse mAb (#2524), NF-κB p65 rabbit mAb (#8242), p21 Waf1/Cip1 rabbit mAb (#2947), and β-Actin mouse mAb (#3700) were purchased from Cell Signaling (Danvers, MA). Alexa Fluor-594 labeled goat anti-mouse IgG (#A10680) and Alexa Fluor-647 labeled goat anti-rabbit IgG (#A21244) were purchased from Thermo Fisher (Waltham, MA). Mouse anti-human p16 mAb (cat# 51–1325GR) was obtained from BD Biosciences. Mouse anti-human CD68 mAb (Cat# 14–0688-82, clone: KP1) was obtained from Thermo Fisher. Pro-Long Gold Anti-Fade Reagent (#9071) was obtained from Cell Signaling Technology (Danvers, MA).

Western blots were performed as previously described (12 (link)) using the following antibodies: pan-actin (clone
Ab-5, Thermo Scientific), fibronectin, vimentin, ZO-1 (BD Biosciences) and
brachyury (MAb 54-1). For immunofluorescence analysis, tumor cells grown on
glass coverslips were rinsed with PBS and fixed for 10 minutes using 3%
paraformaldehyde (Electron Microscopy Sciences). Following permeabilization
using 0.05% Triton X-100 in PBS, coverslips were blocked in PBS
supplemented with 1% BSA and 10% goat serum, incubated with
anti-ESR1 antibody (GTX62423, GeneTex) overnight at 4°C, and
subsequently stained with AlexaFluor-647 labeled goat anti-rabbit IgG (Thermo
Fisher Scientific) for one hour at room temperature, according to the
manufacturer's instructions. Nuclei were stained using DAPI; where
indicated, coverslips were stained with phalloidin Alexa Fluor-488 (Thermo
Fisher Scientific). Images were captured using a Leica Fluorescent microscope
(Leica Biosystems Inc.).