Anti rabbit fluorescein conjugated secondary antibody

Dulbecco’s Modified Eagle’s Medium (DMEM; 12800017), Penicillin-Streptomycin, Trypsin-EDTA (0.05%) and Lipofectamine 2000 were obtained from Gibco (Invitrogen, Carlsbad, CA); fetal bovine serum (FBS) and horse serum (HS) from Natocor (Córdoba, Argentina); cisplatin from Microsules (Buenos Aires, Argentina); and temozolomide and ovine prolactin from Sigma (St. Louis, MO). OCT medium for frozen sections was obtained from Biopack (Buenos Aires, Argentina). Ketamine was obtained from Holliday (Argentina Poniente, Mexico). Xylazine (Kensol) was obtained from König (Buenos Aires, Argentina). Ketoprofen (Ketofen) was from Merial Laboratories S.A. (Buenos Aires, Argentina). Anti-rat PRL is from Dr. A. Parlow, National Hormone and Pituitary Program (NHPP; Torrance, CA). The PRLR antagonist ∆1–9-G129R-hPRL was produced by recombinant technology and purified by ion exchange chromatography as previously described^{17 (link)}. Anti-human PRL (A0569) was obtained from Dako (Santa Clara, CA) and anti-human PRLR antibodies for WB and immunocytochemistry (H-300 and D-7) are from Santa Cruz Biotechnology (Dallas, TX). Anti-rabbit IgG and anti-rabbit fluorescein-conjugated secondary antibody are from Vector Laboratories Inc. (Burlingame, CA). Anti-guinea pig rhodamine-conjugated secondary antibody is from Chemicon International (Temecula, CA).

To assess activation levels, cells were detached, washed and resuspended in PBS, and 1 × 10⁵ cells in 100 μl PBS were stained with antibodies using a 4-color combination as specified in Tables 1 and 2. Samples were incubated for 15 min in the dark and the excess of antibodies was washed with PBS at 300 g for 10 min. Prior to acquisition, 5 μl of 7-aminoactinomycin D (7-AAD) (Becton Dickinson Bioscience. San Jose, CA, USA) was added. A total of 50000 events were acquired in a FACSCantoII (Becton Dickinson Bioscience, San Jose, CA, USA) and the percentage of activated cells in the live cell population was gated using the Flow Jo software program (FlowJo LLC, OR, USA). For cell death assays, the Annexin V PE/7-AAD Kit from BD Pharmingen was used. Cells were stained with 5 μl of Annexin V and 5 μl of 7-AAD in combination to quantify the percentages of apoptotic (Annexin V+/7-AAD-) and necrotic (AnnexinV+/7-AAD+) cells. For intracellular, cleaved caspase-8 staining, bone marrow monocytes were washed with PBS and fixed with PFA and permeabilized with 90% methanol. Cells were washed and blocked (0.5% BSA in PBS), and labeled with anti-cleaved Casp8 antibody (Asp387) (Cell Signalling, Danvers, MA, USA) (D5B2) (1:800) and a secondary anti-rabbit fluorescein conjugated secondary antibody (Vector Laboratories, Burlingame, CA, USA).