Dako diluent

For HepG2 cell immunofluorescence (IF) microscopy, cells were plated on poly L-lysine coated glass coverslips and fixed after treatment with dimethyloxaloylglycine (500 μM) or Compound 3 (50 μM) (Sigma-Aldrich, St. Louis, MO, USA) using 4% paraformaldehyde. Cells were permeabilized using 0.25% TRITON X-100 in phosphate-buffered saline (PBS) × 10 minutes and blocked with Background Sniper (Biocare Medical, Pacheco, CA, USA). Primary antibodies diluted in Dako Diluent against phosphorylated IGFBP-1 at Ser101 (1:400), Ser119 (1:400), or Ser169 (1:200), polyclonal IGFBP-1 (1:2500, a kind gift from Dr. R. Baxter, Sydney, Australia), or PKCα (1:250) and incubated overnight at 4°C. The Alexa Fluor fluorescent secondary antibodies used were diluted in Dako Diluent (1:400) anti-mouse 488 or anti-rabbit 568 (Thermo Fisher Scientific, Waltham, MA, USA) and incubated for 45 minutes at room temperature. Samples were counterstained with DAPI (1:300). The stained coverslips were mounted with ProLong Gold Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA).

For whole mount staining of mammary glands, inguinal gland #4 or #9 were removed, fixed in Carnoys fixative at 4°C overnight, re-hydrated and stained with Carmine Red. Tumors and lungs removed from the mice were fixed in 10% buffered formalin and embedded in paraffin. To quantify metastatic foci in the lungs, each lobe was sectioned and stained with hemotoxylin and eosin (H&E). Image J software was used to quantify the metastatic area in each lobe of the lung. For H&E and IHC staining, 5um sections were cut, deparaffinized in xylene and rehydrated with graded ethanol series. The tumor and lung sections were then stained with either α-F4/80 (Invitrogen MF-48004) or α-Ki67 (Pharmingen 550609) antibodies as described below. All primary and secondary antibodies were diluted in DAKO diluent (DAKO). Sections were blocked with M.O.M. blocking reagent (Vector Labs) for 30min and incubated with primary antibody for 30min. The sections were rinsed and incubated with a biotinylated secondary antibody for 15min. Following the biotinylated secondary antibody, the streptavidin-Alexa dye conjugate was applied for 15min. Sections were washed for 5min with Phosphate-buffered saline (PBS) before incubating with DAB for 2min. Sections were washed in water and counter stained using Meyers hematoxylin followed by a brief rinse in DI water and mounted using Gel/Mount (Biomed, Foster City, CA).

Formalin-fixed, paraffin embedded tissues were sectioned at 5 microns and placed on charged slides. Prepared slides were then deparaffinized in xylenes and rehydrated through graded concentrations of ethanol. Antigen Masking Solution (Vector Labs, Burlingame, CA) was used to regain antigenicity. Slides were blocked for 30 minutes (Dako Serum Free Block, (Dako, Santa Clara, CA) and then incubated in primary antibody, Anti-Estrogen Receptor alpha (AB75635, ABCAM Cambridge, MA) for one hour. The primary antibody was diluted 1:100 with Dako Diluent (Dako, Santa Clara, CA). Negative and positive control slides were included. Negative control slides were incubated in diluent. Slides were rinsed with PBS (Sigma, St Louis, MO) and incubated for 30 minutes with biotinylated goat anti rabbit secondary antibody diluted 1:200 (Vector, Burlingame, CA). Slides were rinsed and then incubated for 30 minutes with Elite ABC Kit (Vector, Burlingame, CA). Slides were developed with DAB substrate (Sigma, St. Louis, MO) for 90 seconds and counterstained in Mayer’s Hematoxylin (Sigma, St. Louis, MO). All incubations were performed in a humidity chamber at room temperature.