Mouse anti human β catenin

This study utilized mouse IgG_2a anti-human PECAM clone hec7 (30 (link)), and mouse IgG_2a anti-human VE-cadherin clone hec1 (31 (link)), all produced in the laboratory via hybridoma methodologies. Mouse anti-human β-catenin was obtained from BD Biosciences (San Jose, California). Rabbit anti-human calreticulin and rabbit anti-EEA1 were obtained from EMD Millipore (Billerica, Massachusetts). Chicken anti-human plakoglobin was a generous gift from Dr. Kathleen Green (32 (link)). Chicken anti-human vimentin for use in immunofluorescence microscopy was obtained from Covance (Princeton, New Jersey) while mouse anti-human vimentin clone v9 for use with western blotting and rabbit anti-human caveolin-1 were obtained from Abcam (Cambridge, Massachusetts). Rabbit anti-human Kinesin 5B was purchased from Sigma-Aldrich (St. Louis, MO). Rabbit anti-human Rab5A (S-19), rabbit anti-human IQGAP1, mouse anti-human LAMP2 and mouse anti-human transferrin receptor were obtained from Santa Cruz Biotechnology (Santa Cruz, California). Goat-anti-mouse and goat-anti-rabbit conjugated to HRP were purchased from BioRad Laboratories. Secondary antibodies (Alexa Fluor 488 or Rhodamine Red-X conjugated to either goat anti-mouse, goat anti-rabbit, or goat anti-chicken) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).

Paraffin sections (4 μM) were deparaffinized and stained with Hematoxylin and Eosin. For immunostaining, sections were deparaffinized in xylene, re-hydrated in a decreasing ethanol gradient, and antigen-retrieval was performed using a de-cloaking chamber at 120°C for 30 seconds and 90°C for 10 seconds in Dako target-retrieval solution followed by blocking with 5% goat serum. To stain mouse sections, rabbit anti-mouse β-catenin antibody (Santa Cruz Biotechnology) (1:100 dilution) was used overnight at 4°C. Antibody binding was revealed by HRP anti-rabbit secondary antibodies (1 hour at room temperature) and counterstained using Gill’s II hematoxylin. Human specimens were stained with mouse anti-Human β-catenin (BD Biosciences) and Rabbit anti-Human CD3 (Neomarkers), or Mouse IgG1 (Abcam) and Rabbit IgG (Abcam) overnight (in Dako antibody-diluent) at 4°C. Sections were then washed 2X with Dako wash buffer for 5 minutes each and incubated with anti-Mouse Alexa Fluor 660 and anti-Rabbit Alexa Fluor 488 (secondary antibodies) for 1 hour at room temperature in the dark. Sections were washed 2X with Dako wash buffer 5 minutes each, stained with DAPI for 10 min, and then washed in PBS and mounted using Gelvatol. Sections were imaged using confocal microscopy (Leica). Images were recorded using the automated TissueGnostic method, and quantified by ImageJ in an unbiased manner.