Dab chromogen

To investigate the expression of Gα_i proteins in human pituitary tumors, Gα_i-1, Gα_i-2 and Gα_i-3 immunostainings were performed in four prolocatinomas, six somatotropinomas, three ACTH and four NFPA tumors. All tumors were AIP mutation negative. Antibodies used were mouse monoclonal antibody against Gα_i-1 (SPM397, sc-56536, Santa Cruz, 1: 40), rabbit polyclonal antibody against Gα_i-2 (T19, sc-7276, Santa Cruz, 1: 60) and mouse polyclonal antibody against Gα_i-3 (H00002773-B01P, Abnova Corp. Taipei city, Taiwan, 1: 50). Anti-mouse/rabbit/rat secondary antibody, Poly-HRP-GAM/R/R (DPVB55HRP, Immunologic, Duiven, Netherlands) and DAB chromogen (Lab Vision Corporation, Fremont, CA, USA, Thermo Fisher Scientific, Watham, MA, USA) were used for detection. Immunostaining protocol was applied as described [30] (link). The staining intensities of Gαi proteins were scaled as negative (0), weak (1), moderate (2), or strong (3). The images were taken and edited by Leica DM LB microscope (Meyer Instruments, Houston, TX, USA), Olympus DP50 camera (Olympus Corporation, Tokyo, Japan) and Studio Lite software (Licor, Lincoln, NE, USA).

Excised tumor specimens were fixed in 10% neutral-buffered formalin. After embeded in paraffin, a series of 5-μm sections were sliced. Sections were de-paraffinized, and rehydrated. After the sections were de-waxed by xylene and processed by graded ethanol debenzolization. They were then washed with tap water, differentiated with hydrochloric acid and ethanol before staining with eosin for 2 min. For Immunohistochemistry analysis, after quenching endogenous peroxidase activity and blocking nonspecific binding sites, slides were incubated at 4°C overnight with 1:100 dilutions of primary antibodies against Bax, Bcl-2, caspase 3, Ki-67 and α-SMA respectively, followed by 30-min incubation with secondary antibodies. Slides were then visualized using the DAB chromogen (Lab Vision Corp., Fremont, CA).