Non specific polyclonal rabbit antibody

The human head and neck tissue microarrays (HN802 and HN803c) were purchased from US Biomax (Rockville, MD). IHC of the human tissue microarrays and paraffin-embedded xenografts was conducted as previously described [16 (link)]. Human tissue microarrays were immunostained with anti-ATAD3A antibody, and sections of tongue tumor xenografts were immunostained with antibodies against phospho-ERK1/2 and Ki67. Negative controls included non-specific polyclonal rabbit antibody at 2 μg/ml (Abcam, Cambridge, MA). Immuno-reactivity was visualized using the DAB Kit (Vector Laboratories, Burlingame, CA) and counterstained with hematoxylin according to the manufacturers’ procedure. Five areas of each sample from xenograft sections or tissue microarrays were imaged at random by a CCD camera (Zeiss), and IHC staining intensity of targeted protein was quantified using Image pro-Plus6.0 software (Media Cybernetics, Silver Springs, MD) and presented as integrated optical density (IOD).

Sections of tongue tumors were immunostained with antibodies against phospho-Src, phospho-AKT, phospho-S6, and Ki67 as described previously [3 (link), 5 (link)]. Negative controls included non-specific polyclonal rabbit antibody at 2 μg/ml (Abcam, Cambridge, MA). The sections were developed with the diaminobenzidine tetrahydrochloride (DAB) substrate kit (Vector Laboratories) and counterstained with hematoxylin.

Sections of paraffin-embedded mouse tongue tumors derived from HN12 cells were deparaffinized and rehydrated by standard pathology laboratory methods, followed by heat-induced antigen retrieval and IHC with antibodies against p-AKT (1:500), p-S6 (1:500) and CD31 (1:800). Negative controls included non-specific polyclonal rabbit antibody at 2 μg/mL (Abcam, Cambidge, MA, USA). The sections were developed with the diaminobenzidine tetrahydrochloride (DAB) substrate kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s recommendations and counterstained with hematoxylin. At least five areas for each sample were imaged at random by a CCD camera (Zeiss, Oberkochen, Germany), and IHC staining of the indicated antibody was quantified using Image pro-Plus6.0 software (Media Cybernetics, Silver Springs, MD, USA) and presented as integrated optical density (IOD).