Mouse anti p120 catenin

Primary antibodies used include mouse anti-p120-catenin (1:500, BD Biosciences 610134), mouse anti-E-cadherin (1:200, BD Biosciences 610182) rat anti-CK8 (1:125, Developmental Studies Hybridoma Bank, Troma-1), rabbit anti-CK14 (1:10.000, Covance, PRB-155P), guinea pig anti-vimentin (1:400, Fitzgerald, 20R-VP004), rabbit anti-δ-catenin (EMD-millipore, 07–259), guinea pig anti-ARVCF (1:100, previously used in [33 ]), guinea pig anti-p0071 (1:100, previously used in [33 ]).
Secondary antibodies used were rabbit anti-guinea pig (DAKO, p0141), HRP conjugated rabbit anti-rat (DAKO p0450), poly HRP anti-rabbit (Immunologic, DPVR500HRP), poly HRP anti-mouse/rabbit/rat (Immunologic, DPVO500HRP), Alexa568 conjugated anti-mouse (1:500, Invitrogen, A11031) and Alexa488 conjugated anti-rabbit (1:500, Invitrogen, A11034).

Cells were grown on glass cover-slips, washed with Ca²⁺- and Mg²⁺-containing PBS and fixed with 4% paraformaldehyde in PBS for 5 min at room temperature. Cells were permeabilized for 3 min using 0.3% Triton X-100 in PBS and blocked in 2% normal goat serum in PBS for 10 min. Blocking was performed by incubation in 10% normal goat serum in PBS for 10 min. Fixed samples were incubated overnight at 4 °C with primary antibodies in 1% bovine serum albumin in PBS using the following antibodies: mouse anti-p120-catenin (1:500; 610134, BD Biosciences) and Alexa Fluor 555-conjugated anti-E-cadherin (1:100; 560064, BD Biosciences). Goat anti-mouse-Alexa Fluor 488 (A11029, Invitrogen) was used as a secondary antibody and incubated for 1 h at room temperature. Samples were stained with DAPI for 3 min and mounted using Immu-Mount (Thermo Scientific). Samples were imaged using a Zeiss LSM 700 microscope (Carl Zeiss) and processed using ImageJ (National Institutes of Health) and Photoshop CS6 (Adobe).