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Background: Breast carcinoma in situ (CIS) is classified into ductal carcinoma in situ (DCIS) and lobular carcinoma in situ (LCIS). DCIS is treated with surgical excision while LCIS can be clinically followed with or without hormonal treatment. Thus, it is critical to distinguish DCIS from LCIS. Immunohistochemical (IHC) staining for E-cadherin is routinely used to differentiate DCIS from LCIS in diagnostically challenging cases. Circumferential diffuse membranous staining of E-cadherin is the typical pattern in DCIS, whereas LCIS lacks or shows decreased E-cadherin expression. Recent studies have shown that DCIS has membranous staining of P120 catenin and LCIS has diffuse cytoplasmic staining of P120 catenin. We developed a cocktail composed of E-cadherin and P120 catenin primary antibodies so that only one slide is needed for the double immunostains., Designs: Twenty-seven blocks of formalin-fixed paraffin-embedded tissue from 26 cases of DCIS or LCIS were retrieved from the archives of Houston Methodist Hospital. Four consecutive sections from the same blocks were used for H&E and immunohistochemical (IHC) stains. The E-cadherin antibody was a rabbit polyclonal antibody and the P120 catenin antibody was a mouse monoclonal antibody. The E-cadherin primary antibody was detected using a secondary antibody raised against rabbit antibody and was visualized with a brown color. The P120 catenin primary antibody was detected using a secondary antibody raised against mouse antibody and was visualized with a red color., Results: Using individual antibodies, 15 of 15 DCIS lesions had diffuse circumferential membranous E-cadherin staining (brown stain) or P120 catenin staining (red stain). All 12 LCIS cases showed cytoplasmic P120 red staining or loss of E-cadherin staining when the single P120 catenin or E-cadherin antibody was used. When stained with the antibody cocktail, all 15 DCIS samples showed diffuse red and brown membranous staining without cytoplasmic stain; all 12 LCIS samples showed diffuse cytoplasmic red staining for P120 catenin but no membranous staining for E-cadherin., Conclusions: 1. This antibody cocktail can be applied in daily practice on paraffin-embedded tissue and is especially useful in small biopsies with small foci of CIS lesions. 2. Immunohistochemical staining with the antibody cocktail showed 100% concordance with the traditional single antibody immunostaining using either E-cadherin or P120 catenin antibody. 3. Our antibody cocktail includes E-cadherin as a positive membranous stain for DCIS and P120 catenin as a positive cytoplasmic stain for LCIS, which may enhance accuracy and confidence in the differential diagnoses.