Alexa546 conjugated anti rabbit igg

MEF or HeLa cells were fixed with 4% (w/v) ultra-pure electron microscopy-grade paraformaldehyde for 20 min at 37 °C and permeabilized with 0.2% Triton X-100 for 10 min at room temperature. The cells were subsequently blocked with 5% (w/v) BSA and Block Ace Powder (DS Pharma Biomedical Co., Ltd., Osaka, Japan) in PBS for 1 h at room temperature, followed by incubation with a combination of 2 of the following antibodies for 1 h at room temperature: anti-p80 antibody23 (link), anti-NuMA antibody (Bethyl Laboratories, Montgomery, USA), anti-γ tubulin antibody (Sigma), and anti-β tubulin antibody (Abcam). The samples were washed off with PBS, and subsequently incubated with Alexa 488-conjugated anti-mouse IgG or Alexa 546-conjugated anti-rabbit IgG (Thermo Fisher Scientific, MA, USA) that contained 400 nM 4′,6-diamidino-2-phenylindole (DAPI) for 1 h at room temperature. Slides were mounted in FluoSave Reagent (EMD Millipore, Darmstadt, Germany), and images were obtained with a laser scanning confocal microscope (LSM700, Carl Zeiss, Oberkochen, Germany).

Sa3 cells were cultured on sterile 18-mm round coverslips. After reaching 70−80% confluence, the cells were treated with 5 mg/mL 5-FU for 1 or 3 h. For immunocytochemistry, the cells were fixed with 3.0% formalin for 30 min and then permeabilized with 0.1% Triton X-100 in PBS for 2 min at 4°C. After blocking of nonspecific binding sites, cells were incubated with anti-NF-κB antibody (sc-372) (Santa cruz) diluted 1∶200 in 4% BSA in PBS overnight at 4°C, followed by Alexa 546-conjugated anti-rabbit IgG (Molecular Probes, Eugene, OR, cat no A11010), diluted 1∶500 in 4% BSA in PBS for 60 min at ambient temperature. The samples were mounted and examined under a microscope equipped with epifluorescence illumination (ECLIPSE Ti-U, Nikon). To exmamine the effect of NF-κB on 5-FU-suppressed cellular viability, Sa3 cells incubated with NF-κB inhibitors, 5 µM BAY 11-7085 (Enzo Life Sciences, cat no EI-279) and 200 µM Caffeic acid phenethyl ester (CAPE) (Calbiochem, San Diego, CA, cat no 211200) for 1 h. Then cells were treated with 5 mg/mL 5-FU for 24 h and cellular viability was mesured by WST-8 assay.

Preparation of PAG sections (n = 3) after FG injection into the MRF and the procedure until blocking with 1.5% blocking solution is described in the section on ISH for vGLUT2 mRNA. After blocking, the sections were incubated with a mixture of sheep horseradish peroxidase-conjugated anti-DIG antibody (1:20, Roche Diagnostics Corp.) and rabbit anti-FG antibody (1:1000, Invitrogen) overnight at RT. Then the sections were incubated for 30 min in biotin-conjugated tyramide (1:50 in amplification diluent, PerkinElmer, Waltham, MA). Following several washings, the sections were incubated with a mixture of Alexa 488-conjugated streptavidin and Alexa 546-conjugated anti-rabbit IgG (1:500, Molecular Probes, Eugene, OR) for 2h at RT.

Rats were injected with tribromoethanol (200 mg/kg, i.p.) for anesthesia, and transcardially perfused with isotonic 0.1 M phosphate buffer (pH 7.4), followed by isotonic 4% PFA. Then, the brain was fixed in 4% PFA at 4°C overnight and cryoprotected in 20–30% sucrose in 0.1 M phosphate buffer. Briefly, sections (20 μm) were fixed with 4% PFA and washed with 0.3% Triton X-100/PBS. Subsequently, the sections were incubated for 1 h in blocking serum (5% normal donkey serum in 0.2% Triton-X 100/PBS) at room temperature and then with primary antibodies (anti-ASIC1, Alomone Labs, Jerusalem, Israel; anti-Parvalbumin, Swant, Marly1, Switzerland) at 4°C overnight. Finally, the sections were washed with PBS and incubated with species-matched secondary antibodies (Alexa 488-conjugated anti-mouse IgG; Alexa 546-conjugated anti-rabbit IgG, Invitrogen) at room temperature for 1 h. The images were captured using the Evos FL auto 2 microscope (Invitrogen).

Anti-Aurora B (ab2254), anti-INCENP (ab36453) and anti-HA (16B12; ab130275) antibodies were obtained from Abcam. Anti-Plk pT210 (sc-135706) and normal rabbit IgG were obtained from Santa Cruz Biotechnology. Anti-Cdc7 antibody was obtained from MBL. Anti-Aurora B pT232 antibody (Poly6361) obtained from BioLegend was used only for immunostaining. Anti-Histone H3 pS10 rabbit antibody was from Upstate. Anti-Histone H3 pS28 antibody was prepared in house in rat^{70 (link)}. Anti-CENP-A pSer7 antibody (2187) was from Cell Signaling Technology. Antibodies against Tubulin, Aurora-A and FLAG (M2) were obtained from Sigma. Alexa 488 conjugated anti-rabbit IgG, Alexa 546 conjugated anti-rabbit IgG, Alexa 488 conjugated anti-rat IgG, and Alexa 647 conjugated anti-rabbit IgG, obtained from Invitrogen, were used for immune-staining or FACS staining.

HEK293 cells were transfected with pEGFP/flotillin-1-GFP plus pcDNA3.1(+)/ABCG1, pcDNA3.1(+)/ABCG1-K120M, pcDNA3.1(+)/ABCG4, or pcDNA3.1(+)/ABCG4-K120M. After 24 h of transfection, cells were subcultured on glass cover slips treated with poly-L-lysine (Sigma). After 48 h of transfection, cells were either fixed with cold methanol or with 4% paraformaldehyde in PBS+(phosphate-buffered saline containing 0.1 g/l of CaCl₂ and MgCl₂·6H₂O), and permeabilized with 0.4% TritonX-100 in PBS+ for 5 min. To reduce nonspecific binding of antibodies, the cells were incubated in 10% goat serum in PBS+. Then, the cells were incubated for 1 h with a rabbit polyclonal anti-ABCG1 or anti-ABCG4 antibody, and then incubated with fluorescent Alexa546-conjugated anti-rabbit IgG (Invitrogen) for 1 h. Cells were directly visualized with a 63x Plan-Neofluar oil immersion objective using a Zeiss confocal microscope (LSM700).