Alexa fluor 555 donkey anti rabbit igg h l

Ovulated eggs were fixed in 4% paraformaldehyde in PBS (pH 7.4) for 30 minutes and permeabilized in 0.5% Triton-X-100 for 20 min at room temperature. Then, oocytes were blocked with 1% BSA-supplemented PBS for 1 h and incubated at 4°C overnight or at room temperature for 4 h with rat monoclonal anti-mouse folr4 antibody (1:100, BioLegend, CA) or rabbit polyclonal anti-mouse ovastacin antibody (1:100, obtained from Dr. Jurrien Dean). After washing four times (5 min each) in PBS containing 1% Tween 20 and 0.01% Triton-X 100, eggs were incubated with an appropriate secondary antibody for 1 h at room temperature. Alexa Fluor 555 donkey anti-rabbit IgG (H+L) was obtained from Invitrogen (Carlsbad, CA). After washing three times, eggs were stained with PI or Hoechst 33342 (10 μg/ml) for 10 min. Finally, eggs were mounted on glass slides and viewed under a confocal laser scanning microscope (Carl Zeiss 700).

The parasites were embedded in OCT compound and serial cryosections (5–7 μm) were obtained. The tissue sections were fixed for 10 min in 4% formaldehyde and rinsed with PBS containing 0.3% Triton X-100, and were then incubated in blocking solution (5% bovine serum albumin in PBS) for 2 h at 25°C, followed by rabbit polyclonal antibodies against rSjTH (2 mg/mL, 1:500 dilution) and IgG from a non-immunized rabbit in blocking solution overnight 4°C. Parasite tissue sections were further incubated with Alexa Fluor 555 donkey anti-rabbit IgG (H+L) and 4’, 6-diamidino-2-phenylindole (DAPI, all from Invitrogen, Waltham, MA, USA). Fluorescence was visualized using a TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany).

A rabbit polyclonal antibody that binds a C-terminal peptide of ovastacin^395-408 and monoclonal antibody M2c.2 that binds to the C-terminal region of ZP2 have been characterized previously [32 (link)][45 (link)]. The following antibodies and lectins were obtained commercially: LCA-FITC (Sigma-Aldrich); antibodies to GP73, calregulin and EEA1, Alex Fluor 488 goat anti-rabbit IgG (H+L)(Life Technologies-Invitrogen, Carlsbad, CA); Alexa Fluor 555 donkey anti-rabbit IgG (H+L) (Life Technologies-Invitrogen); DyLight 649 goat anti–rabbit IgG (H+L) (Life Technologies-Invitrogen); and goat anti-rat IgG-HRP (Santa Cruz).

Deciduous teeth were collected from 3- to 11-year-old patients (n = 3) and permanent teeth were collected from adult patients (19 to 81 years of age, n = 3). Teeth were cracked open and pulps were collected and snap frozen in optimal cutting temperature (OCT) cryomount (Histolab, Gothenburg, Sweden) for subsequent 5 μm sectioning. The sections were fixed for 20 minutes in 4% paraformaldehyde (Merck, Darmstadt, Germany), rinsed and then incubated overnight at room temperature with primary antibodies against HMGA2 (1:500, Cell Signaling Technology) and vimentin (1:200, Abcam). Additional staining was performed the following day with 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen, Burlington, Canada) at 300 nM applied for three minutes. Secondary antibodies used were Alexa Fluor® 555 Donkey Anti-Rabbit IgG (H + L) and Alexa Fluor® 488 Goat Anti-Chicken IgG (H + L) (Invitrogen, Eugene, OR, USA). Control sections were incubated where the primary antibody was omitted. This resulted in no detectable artifact. Additional negative control stainings were performed with isotype control for rabbit primary antibody (Invitrogen, Eugene, OR, USA), which resulted in no unspecific staining. Confocal microscopy was performed with Zeiss LSM700 CLSM and image processing was carried out with Imaris software.

Immunofluorescent analyses were performed on 4-μm frozen sections of free islets (24 hours post treatments) or of livers transplanted with islets (30 days after graft). The angiogenic properties of liraglutide were determined using a mouse anti-rat cluster of differentiation 31 (CD31) antibody (1/200; BD Biosciences^™, Le Pont de Claix, France) and the insular structure of the islets was examined using a rabbit anti-rat insulin antibody (1/100, Cell Signaling). The appropriate secondary antibodies (Alexa Fluor^® 555 donkey anti-rabbit IgG [H+L] [1/1000, Invitrogen, Fisher, Ilkirch, France] and Alexa Fluor^® 488 donkey anti-mouse IgG [H+L] [1/1000, Invitrogen]) were used to visualise these signals and the appropriate positive and negative controls were performed. Fluorescence intensity was measured by microscopy and analysed by NIS-Elements Br Software (Nikon Instruments Inc., Champigny-sur-Marne, France). Ten different islets per condition were measured and data were expressed as the mean value of fluorescence intensity reported to analysed surface ± the standard error of the mean (SEM).

Mouse femurs and tibiae were cleaned of muscle, fixed in 4% paraformaldehyde (MilliporeSigma) for 8 hours, and dehydrated in 30% sucrose solution for 24 hours. Bones were sectioned on a Leica CM1950 microtome and incubated in staining buffer (20% DMSO [MilliporeSigma], 5% donkey serum [Jackson ImmunoResearch], and 0.5% IGEPAL [MilliporeSigma]) in PBS containing primary antibodies for 8 hours. After washing with PBS and gentle shaking for 4 hours, the sections were incubated with secondary antibodies for 8 hours in the staining buffer mentioned above. After washing with PBS and gentle shaking for a further 4 hours, a rabbit anti-perilipin (MilliporeSigma, 1:400) primary antibody was used to label adipocytes, which were detected with a donkey anti–rabbit IgG (H+L) Alexa Fluor 555 (Thermo Fisher Scientific, 1:400) secondary antibody. DAPI was used to label all nucleated cells. Images were obtained using 2-photon excitation microscope (Olympus). 3D reconstruction was created using Velocity software (PerkinElmer).