Biotinylated anti rabbit igg h l

IHC was performed on a Leica Bond RX automated stainer using Bond reagents (Leica Biosystems, Buffalo Grove, IL), including a polymer detection system (DS9800, Novocastra Bond Polymer Refine Detection, Leica Biosystems). The chromogen was 3,3 diaminobenzidine tetrachloride (DAB), and sections were counterstained with hematoxylin. Staining for cleaved caspase 3 (CC3) used primary antibody (Cell signaling, Cat.9961) at 1:250 dilution and biotinylated anti-Rabbit IgG (H+L) as secondary antibody (Vector, Cat. BA1000) at 1:100 dilution. Staining for mouse NKp46 used primary antibody (R&D Systems, Cat. AF2225) at 1:1000 dilution and biotinylated anti-Goat IgG (H+L) (Vector, Cat. BA5000). Both markers used heat induced, pH 6.0 for epitope retrieval.

Samples were deparaffinized and rehydrated. Antigen was retrieved using 0.01 M sodium-citrate buffer (pH 6.0) at a sub-boiling temperature for 10 min after boiling in a microwave oven. To block endogenous peroxidase activity, the sections were incubated with 3% hydrogen peroxide for 10 min. After 1 h of preincubation in 5% normal goat serum to prevent nonspecific staining, the samples were incubated with the antibody to IκBα (1:50, Cell Signaling Technology, 4814) or TNFα (1:50, Novus Biologicals, NBP1-19532) at 4°C overnight. The sections were incubated with a biotinylated secondary antibody (1:500, biotinylated anti-rabbit IgG(H+L), Vector Laboratories, BA-1000 for TNFα; 1:500, biotinylated anti-mouse IgG(H+L), Vector Laboratories, BA-9200 for IκBα) and then incubated with an avidin-biotin peroxidase complex solution (Vector Laboratories, PK-6100) for 30 min at room temperature. Color was developed using the DAB (diaminobenzidine) Substrate Kit (BD Biosciences, 550880). Counterstaining was carried out using Harris modified hematoxylin.

Portions of tumors were fixed overnight in 10% neutral buffered formalin, and processed into 5 μm sections for immunostaining [25 (link)]. Sections were counterstained with hematoxylin and imaged using an E600 Eclipse microscope with NIS-Elements imaging software. The following antibodies were used: ERα (1/500; Santa Cruz Biotechnology, #SC-542), pAKT^S473 (1/50; Cell Signaling Technologies, #3787), YAP (1/400; Cell Signaling Technologies, #14074), cleaved caspase-3 (Asp175) (1/100; Cell Signaling Technologies, #9661), pp70S6K (1/100; Cell Signaling Technologies, #9205), Ki-67 (1:500; Abcam, #15580), pS6RP^S235/236 (1/400; Cell Signaling Technologies, #4858), and biotinylated anti-rabbit IgG (H&L) (Vector Laboratories, #BA-1000). Labeled cells were quantified by counting 200 cells in each of five random 40x fields of view (FOV) for each tumor.
All lungs from tumor-burdened mice were processed as above, and metastatic load was quantified in three step-sections, 50 μm apart. Metastases in ten random 10x FOVs were counted in each section, taking care to avoid recounting metastases which spanned multiple step-sections. Lesion area was quantified by tracing freehand perimeters using ImageJ software [26 ].