Alexa fluor 594 goat anti rabbit secondary antibody

Cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, permeabilized, and stained in a blocking solution containing the antibodies, 0.03 g/ml bovine serum albumin (BSA, Sigma), 10% goat serum (Sigma), and 0.3% Triton X-100 (Sigma) in PBS for 2 hr. The samples were washed before incubation with secondary antibodies for 1h at RT. The samples were then mounted in Fluoro-Gel (Electron Microscopy Sciences, Hatfield, PA, USA) for fluorescent imaging.
Antibodies against the following proteins were applied for immunofluorescence (IF). Monoclonal antibodies: Neuronal class III β-tubulin (Tuj1) 1:500 (Covance, Princeton, NJ), MAP2 1:500 (BD Biosciences, Franklin Lakes, New Jersey). Rabbit polyclonal antibodies: Neuronal class III β-tubulin (Tuj1) 1:500 (Covance). Secondary antibodies used for IF were Alexa Fluor 594 goat anti-rabbit secondary antibody 1:200 (Life Technologies/Invitrogen), or Alexa Fluor 488 goat anti-mouse secondary antibody 1:200 (Life Technologies/Invitrogen) and cell nuclei were stained with 4,6-Diamidino-2-phenylindole (DAPI) 1:5000 (Life Technologies/Invitrogen).

Immunohistochemistry for microglia was performed as described with minor alterations (Campbell et al., 2015 (link)). Briefly, fixed brains were transferred to 30% sucrose, and serial sections (30 µm) throughout the hippocampus were prepared by a sliding microtome (Microm International, Heidelberg, Germany). Sections were blocked in phosphate saline–blocking buffer (1% bovine serum albumin, 0.2% Triton X-100) for 1 hour at room temperature prior to primary antibody incubation. To detect microglia, slices were incubated with an antibody against ionized calcium-binding adapter molecule 1 (Iba-1, 1:1000, Wako Chemicals USA, Inc, Richmond, VA, USA) overnight at 4 °C followed by AlexaFluor 594 goat anti-rabbit secondary antibody (1:2000, Invitrogen) for 1 hour at room temperature. Sections were washed with 1× phosphate-buffered saline with 0.2% Triton X-100. Slices were then incubated with 4′, 6-diamidino-2-phenylindole (1:10,000, Sigma) for 10 minutes at room temperature and then mounted using Fluoro- Gel with TES buffer (Electron Microscopy Sciences, Hatfield, PA, USA). Images were analyzed with a Nikon Eclipse Ni microscope at 450-nm and 594-nm wavelengths.

Ombrabulin (0, 30, or 60 mg/kg) was administered intravenously to mice bearing bilateral flank MDA-MB-435 xenografts. At different time points (4 or 24 hrs post-injection), the mice were euthanized and their tumors were excised in their entirety. The tumors were gently dissociated into single cell suspensions using a MACS human Tumor Dissociation Kit (Miltenyi Biotec) according to manufacturer instructions. 2.5 × 10⁶ cells per condition were incubated for 1 hr on ice with both Alexa Fluor-488-conjugated mouse anti-human HLA-AB (BD Pharmingen) and either rabbit polyclonal anti-p32 (Millipore) or rabbit IgG isotype control (R&D Systems). Cells were then washed twice with cold PBS supplemented with 2% fetal bovine serum (FBS), followed by incubation for 1 hr on ice with Alexa Fluor-594 goat anti-rabbit secondary antibody (Invitrogen). Cells were washed and then resuspended in PBS plus 2% FBS for analysis. For quantification of surface p32 levels, human tumor cells were isolated by gating out all HLA-ABC-negative cells.