Alexa fluor 488 antirabbit igg secondary antibody

On days 7 and 14, the samples were fixed with cold 70% methanol and stored at −20 °C until analysis. After thawing for a few minutes, the samples were washed with PBS and incubated with 1% bovine serum albumin (BSA) in PBS/0.1% (v/v) Triton-X for 30 min at RT. After washing with PBS, the primary monoclonal antibody against type I collagen (dilution 1:200, LB-1197, CosmoBio Co., LTD., Tokyo, Japan) was applied overnight at 2–8 °C. The day after, the samples were incubated with Anti-Rabbit IgG Alexa Fluor^® 488 secondary antibody for 45 min at RT in the dark (Invitrogen, Life Technologies, Eugene, OR, USA). After washing with PBS, cell nuclei were stained for 5 min with propidium iodide (PI) (RT, 5 mg/mL in PBS, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The cells were visualized using a confocal microscope Zeiss LSM 880 Airyscan, (Zeiss, Oberkochen, Germany) at ʎ_exc = 488 nm and ʎ_em = 505–550 nm for Alexa Fluor 488 and at ʎ_exc = 535 and ʎ_em > 570 nm for PI.

The immunofluorescence staining for NF-κB was measured as previous study described [20 (link)]. The cells were cultivated on coverslips and incubated with various concentrations of K36 after UVB exposure. The cells were fixed with 4% paraformaldehyde and blocked with 5% nonfat milk containing 0.3% Triton X-100/PBS. After incubating with the primary antibody, the cells were incubated with the Alexa Fluor 488 antirabbit IgG secondary antibody (Invitrogen, USA). The unbound secondary antibody was removed using PBS. Thereafter, the samples were counterstained with the ProLong Gold antifade reagent with DAPI, and observed using a microscope (Leica DMIL, Germany). The visual scoring of the translocation on each slide was based on the characterization of 100 randomly selected cells.

Explanted cardiac patches were fixed in 4% para-formaldehyde, embedded in paraffin, sliced (5 μm thick sections), and stained with Hematoxylin and Eosin (H&E). H&E staining was used for cell counting. One representative section from each explant at 16 weeks after implantation was stained and imaged. All positively stained nuclei were counted from high magnification images. Collagen deposition was assessed with Picrosirius red staining and images were obtained with polarized light microscopy. Based on previous reports, we correlated thick fibers (orange to yellow) with collagen I and thin fibers (green) with collagen III [11 (link)].
Immunofluorescent staining for α-actinin as a marker of cardiomyocytes was performed using rabbit anti-α-actinin (sarcomeric) primary antibody (1:200, Abcam, Cambridge, MA) followed by Alexa Fluor 488 anti-rabbit IgG secondary antibody (1:300, Invitrogen, Carlsbad, CA). Fluorescent images were obtained with an Olympus IX51 microscope (Olympus, Tokyo, Japan).

Cells were cultivated on glass cover slips as described in a previous paper [53 (link)]. After UVB exposure, various concentrations of CAE (5–25 μg/mL) were added and incubated for 24 h. The cells were then immersed in 4% paraformaldehyde and incubated with nonfat milk containing Triton X-100. A primary antibody was added to the cells, and the cells were then incubated with an Alexa Fluor 488 antirabbit IgG secondary antibody (Invitrogen, Carlsbad, CA, USA). The cells were washed with PBS buffer to rinse off the unbound secondary antibody. Finally, the samples were counterstained with the ProLong^® Gold antifade reagent and viewed using a microscope (Leica DMIL, Wetzlar, Germany).

Infected cells plated on coverslips or parasite pellets were washed with PBS and fixed with PBS–4% paraformaldehyde, permeabilized for 5 min with 0.1% Triton X-100, and blocked for 30 min with 5% bovine serum albumin (BSA) (all reagents purchased from Sigma). Cells from infectivity and replications assays were incubated for 1 h at room temperature with anti-cytosolic tryparedoxin peroxidase (cTXNPx) primary antibody (52 (link)) and for 30 min with Alexa Fluor 488 anti-rabbit IgG secondary antibody (Invitrogen, Eugene, OR). Parasites from the epimastigogenesis experiment were attached to poly-L-lysine-coated slides and treated as monolayers, except for those incubated with a monoclonal primary antibody (MAb25, kindly provided by Sergio Schenkman; 1:1,000 dilution) that binds to the T. cruzi flagellar calcium-binding protein (53 (link)). All antibodies were diluted in 1% BSA/PBS, and coverslips and parasites were mounted with ProLong antifade reagent.

Human neutrophils cultured under the designated condition for 6 h were fixed with 4% paraformaldehyde (PFA) for 10 min and permeabilized with PBS containing 0.2% Tween20 for 10 min. After washing with PBS three times and incubating for 20 min in the SuperBlock (PBS) Blocking Buffer (ThermoFisher Scientific, MA, USA), cells were treated with a rabbit anti-LL-37 antibody (1:1000; OSC00009W, Osenses Pty Ltd., Australia) for 1 h at room temperature. After washing with PBS containing 0.1% Tween20 three times, LL-37 was labeled with Alexa Fluor 488 anti-rabbit IgG secondary antibody (1:1000; Invitrogen Inc., CA, USA) for 1 h at room temperature. After an additional three washes in PBS containing 0.1% Tween20, DNA was stained with DAPI (NucBlue Fixed Cell ReadyProbes Reagent, ThermoFisher Scientific, MA, USA). Slides were mounted in ProLong Gold antifade reagent (ThermoFisher Scientific, MA, USA). Images were acquired with a Zeiss LSM700 confocal microscope (Carl Zeiss, Germany). Separately, for staining of live/dead neutrophils and NETs, Hoechst33342 (2 drops per mL; NucBlue Live Cell ReadyProbes Reagent, ThermoFisher Scientific, MA, USA) and SytoxOrange (1:1000; Invitrogen Inc., CA, USA) were added into the culture medium 6 h after initiation of treatment. Neutrophils were then observed with EVOS FL Cell Imaging System (Life technologies, CA, USA).

Immunofluorescence staining was measured as previously described with some modifications [46 (link)]. The cells were collected, fixed with 4% paraformaldehyde, and blocked with 5% nonfat milk containing 0.3% Triton X-100/PBS. After incubating with the primary antibody, the cells were incubated with Alexa Fluor 488 antirabbit IgG secondary antibody (Invitrogen, USA). The unbound secondary antibody was removed using PBS. Thereafter, the samples were counterstained with the ProLong Gold antifade reagent with DAPI and observed using a confocal laser scanning microscope (Leica DMIL, Munchen, Germany). The visual scoring of the Nrf2 translocation and 8-OHdG expression on each slide was based on the characterization of 100 randomly selected cells. The intensity of fluorescence of Nrf2 was quantified by using Image J.