Alexa 488 anti rabbit secondary antibody

PFA fixed, 30-μm-thick coronal brain slices were obtained and processed for immunofluorescence analysis. Immunofluorescence was performed, after blockade with 5% goat serum, by overnight incubation at 4°C with a GluA1 CTD primary antibody (rabbit, Synaptic Systems, #182–003) followed by incubation with an Alexa 488 anti-rabbit secondary antibody (Invitrogen). Images were obtained using a Leica DMRB fluorescence microscope and processed with ImageJ.

To show the OSNs that express Orco in the third antennal segment, the following fly lines were used: orco-LexA (kindly provided by Benton, R., University of Lausanne, Switzerland) and lexAop-mCD8: GFP (w*; P{y^+t7.7 w^+mC = 13XLexAop2-mCD8::GFP}attP40/CyO, Bloomington Stock Center, Bloomington, IN, USA). Rabbit anti-GFP primary antibody (1:5000; Invitrogen, Waltham, MA, USA) and Alexa 488 anti-rabbit secondary antibody (1:1000; Invitrogen, Waltham, MA, USA) were used to performed an immunofluorescence on antennal cryosections in the lexAop-mCD8: GFP;orco-LexA genotype as previously described [28 (link)].

DU145 tumor cells, grown in covered wells of a microscope slide, were treated with 55 nM DTX for 24 h at 37 °C. Then, the cells were fixed and dually stained with anti-clusterin and anti-HMGB1. Briefly, slides were blocked with goat serum, followed by incubation with mouse anti-human clusterin (Upstate, 1:500) and rabbit anti-human HMGB1 (Cell Signaling, 1:200) overnight at 4 °C. Next day, slides were incubated with an Alexa 549 anti-mouse secondary antibody (Invitrogen, 1:1000) or Alexa 488 anti-rabbit secondary antibody (Invitrogen, 1:1000) for 30 min. The slides were mounted with Everbrite mounting medium with DAPI (Biotium). Slides were analyzed with an automated Zeiss Imager Z.1 upright microscope through a 63×/1.4 NA objective with DAPI, FITC, and Alexa 549 filters. Images were captured by using the AxioCam MRm CCD camera and Axiovision (Version 4.7; Carl Zeiss).

Cells were fixed with 4% formaldehyde, permeabilised with 0.2% Triton X-100 for 5 min, blocked with 5% BSA-PBS for 1 hr at room temperature, and incubated with anti-p-MLC2 (p-MLC2S19, 1:200 in 5% BSA-PBS) overnight at 4°C. Alexa-488 anti-rabbit secondary antibody (Life Technologies) was used at 1:500 for 1 hr at room temperature. F-actin was detected with Phalloidin (1 hr RT) and nuclei were stained with Hoechst 33258 (Life Technologies). Imaging was carried out on a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss) with C-Apochromat × 40/1.2 NA (water) or a Plan Apochromat × 63/1.4 NA (oil) objective lenses and Zen software (Carl Zeiss). Line scan analysis was performed in ImageJ.

Mice peri-implant soft tissue specimens were fixed with 4% formaldehyde, permeabilized with 0.2% Triton X-100 for 5 min, blocked with 5% BSA-PBS for 1 hr at room temperature, and incubated overnight with anti-p-MLC2 (p-MLC2S19, 1 : 200 in 5% BSA-PBS) at 4°C. Alexa-488 anti-rabbit secondary antibody (Life Technologies) at a ratio of 1 : 500 was used for 1 hr at room temperature. Cell nuclei were stained with DAPI (Life Technologies). Imaging was performed on a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss) and analyzed using Zen software (Carl Zeiss).

Preimplantation development, from the mouse 2PN zygote to the blastocyst stage, was evaluated on day 5 after hCG injection [28 (link)]. Developed blastocysts were fixed in 4% paraformaldehyde with 1% polyvinyl alcohol for immunocytochemistry (ICC). The embryos were blocked in 3% BSA (Biosesang, Seongnam, Korea) and incubated with a monoclonal mouse antibody against Oct-4 (Abcam, UK), which is expressed in the inner cell mass (ICM), overnight at 4°C. In ICC of the negative control group, the embryos were incubated without a specific primary antibody against Oct-4. On the next day, blastocysts were conjugated with Alexa 488 anti-rabbit secondary antibody (Life Technology, Norway), and the nuclei were counter stained with 4′,6-diamidino-2-phenylindole (Vector Laboratories, USA). Images were obtained using a fluorescence microscope (AX-70, Olympus, Tokyo, Japan) with IMT i-Solution program (I-solution, British Columbia, Canada). Total blastomere cell number and ICM number were counted with captured fluorescence image, and then, trophectoderm (TE) number was calculated by subtracting the number of ICM from the total cell number [29 (link)].

Immunofluorescence staining of aortic root sections was performed using rabbit anti-mouse GPx1 antibody to observe the intensity of GPx1 in the aortic roots. Briefly, air-dried cryostat sections (6 μm thickness) of aortic root were fixed with the pre-cooled acetone for 10 min and rinsed with 1× phosphate buffered saline (PBS) three times. Non-specific staining was blocked by incubation with 5% goat serum in 1× PBS for 30 min followed by incubation of anti-GPx1 antibody overnight at 4°C in a 1:200 dilution. After rinsing with 1× PBS, sections were incubated with Alexa 488 anti-rabbit secondary antibody (Invitrogen Molecular Probes, UK) for 1 h while nuclei were stained with DAPI in the mounting agent (Invitrogen Molecular Probes). Microscopic examination was performed on an Olympus IX-83 inverted fluorescence microscope (Olympus, Tokyo, Japan). Images were acquired by a monochrome CCD camera using CellSens software (Olympus, Tokyo, Japan).