Alexa fluor 568 goat anti rabbit igg

Mice were sacrificed with overdose of anesthesia. After perfusion, the spinal cord tissue at 0.8–1 cm around the injury site was collected, placed in a gradient sucrose solution to fully submerge, and then 10 μm frozen section was performed.
Before staining, the frozen sections of different groups were washed to remove the OCT embedding agent. After permeabilization with 0.3% Triton X-100 for 15 min, the sections were blocked with goat serum (ZSGB-BIO) for 2 h. Subsequently, serum diluted primary antibodies (anti-NeuN, Mouse, 1:1000, Abcam, ab77315; UHRF1 Antibody, Rabbit, 1:50, Affinity, DF6929) were added and incubated overnight at 4 °C. The next day, the corresponding secondary antibody (Alexa Fluor 488 goat anti-mouse IgG, Thermo Fisher Scientific, A32728; Alexa Fluor 568 goat anti-rabbit IgG, Thermo Fisher Scientific, A-11011, both 1:1000) was incubated at 37 °C 2 h. Finally, stain with DAPI staining solution (Beyotime, C1002) containing anti-fade reagent for 15 min. A coverslip was added, and images were captured using a Leica fluorescence microscope. Cell samples are processed similarly to tissues. Briefly, fix, penetrate, block, incubate primary antibody, secondary antibody, DAPI. Finally, the fluorescence intensity was observed with a Leica confocal laser scanning microscope.

Hemocyte immunofluorescence assays (IFAs) were performed as previously described (14 (link)). Hemolymph perfused from mosquitoes at naïve, 24 h post-blood meal and 24 h post-infection was placed on a multi-well glass slide (MP Biomedicals) and allowed to adhere at RT for 30 min. Cells were fixed with 4% paraformaldehyde for 15 minutes at RT, then washed three times in 1xPBS. Samples were incubated with blocking buffer (0.1% Triton X-100, 1% BSA in 1xPBS) for 24 h at 4°C and incubated with a 1:500 of rabbit anti-AgPGE2R (CVRYRSATEPID: second extracellular loop) affinity-purified antibodies (0.6 mg/ml) (Pacific Immunology), or pre-immune serum (1:1000) in blocking buffer overnight at 4°C. After washing 3 times in 1xPBS, an Alexa Fluor 568 goat anti-rabbit IgG (1:500, Thermo Fisher Scientific) secondary antibody was added in blocking buffer for 2 h at RT. Slides were rinsed three times in 1xPBS and mounted with ProLong^®Diamond Antifade mountant with DAPI (Life Technologies). Images were analyzed by fluorescence microscopy (Nikon Eclipse 50i, Nikon) and confocal microscopy (Leica SP5 X MP confocal/multiphoton microscope) at the Iowa State University Microscopy Facility.

Immunofluorescence studies were performed as previously described [63 (link)]. RBMVEC grown on 12 mm in diameter fibronectin-coated glass coverslips were treated with AMG837 (10 µM, 30 min) and then processed for immunocytochemistry studies; untreated cells served as controls. Cells were first washed with phosphate buffer saline (PBS) and then fixed with 4% paraformaldehyde (20 min, room temperature). Cells were washed in PBS and PBS with 0.5% Triton X for 5 min and incubated in normal goat serum (1:20, 1 h, room temperature). Cells were incubated overnight at 4 °C with the following primary antibodies: ZO-1 (1:200, rabbit polyclonal, Cat # 40–2200, Thermo Fisher Scientific, Waltham, MA, USA) and VE-Cadherin (1:200, rabbit polyclonal, Cat # 36–1900, Thermo Fisher Scientific) followed by incubation with secondary antibody Alexa Fluor 488 goat anti-rabbit IgG (1:200, Cat # A11008, Thermo Fisher Scientific) or Alexa Fluor 568 goat anti-rabbit IgG (1:200, Cat # A11011, Thermo Fisher Scientific) for 2 h at room temperature. Additionally, cells were incubated in ActinRed 555 (Thermo Fisher Scientific) for 30 min at room temperature. Cells were washed in PBS and then mounted with DAPI Fluoromount G (SouthernBiotech, Birmingham, AL, USA) on glass microscope slides. Cells were examined under a Leica DMI6000B fluorescence microscope equipped with the appropriate filters.

Samples were washed with PBS, fixed with 4% paraformaldehyde solution (Wako Pure Chemical Industries) for 20 min, permeabilized with 1% Triton™ X-100 (Sigma-Aldrich) in PBS for 20 min, and then stained overnight with the following antibodies: phycoerythrin (PE) mouse anti-human NANOG, Alexa Fluor^® 488 mouse anti-OCT3/4 (BD Biosciences), mouse anti-TUJ1 (β-tubulin III; Sigma-Aldrich), rabbit anti-α-SMA (PA1-37024, Thermo Fisher Scientific), and rabbit anti-AFP (Abcam, Cambridge, UK). The secondary antibodies Alexa Fluor^® 488 goat anti-mouse IgG (Thermo Fisher Scientific) and Alexa Fluor^® 568 Goat Anti-Rabbit IgG (Thermo Fisher Scientific) were then added. The nuclei were stained with Hoechst 33342 (Lonza).

The immunofluorescence assay was performed to detect the localization of PfAP2-EXP2 as described previously (Jing et al., 2018 (link)). Parasites were harvested, released, fixed by 4% paraformaldehyde (Electron Microscopy Sciences) at room temperature for 10 min, and washed by PBS. Prepared samples were incubated with the primary antibodies against ty1 (Sigma, SAB4800032) or GFP (Abcam, ab290) at 1:500 to 1:1,000, followed by the secondary antibodies AlexaFluor 488 goat anti-mouse IgG (ThermoFisher Scientific, A11029) or AlexaFluor 568 goat anti-rabbit IgG (ThermoFisher Scientific, A11036) at 1:500. Preparations were visualized with a Nikon A1R microscope at 60-100 × magnification and images were acquired with NIS Elements software and processed using Adobe Photoshop.

Cells were plated at 30,000 cells per well on 13 mm round glass coverslips in 24-well plates and transiently transfected as described above. Cells were fixed with pure ethanol for 20 min at −20 °C and blocked with 5% goat serum. The cells were then incubated in primary antibody (JAM-A-sc-53623, Santa Cruz, Heidelberg, Germany; FoxA1-58613, Cell Signaling; β-catenin-8480, Cell Signaling) overnight at 4 °C. Cells were incubated in blocking buffer containing 2 μg/mL secondary antibody (either Alexa-Fluor-488 goat anti-mouse IgG, A32723 or Alexa-Fluor-568 goat anti-rabbit IgG, A11079, ThermoFisher) and then inverted onto slides using Vectashield mounting medium containing DAPI (H-1200, Vector Laboratories, Burlingame, CA, USA). Fluorescent micrographs were acquired at 40× magnification using a CKX41 epifluorescent microscope (Olympus, Hamburg, Germany) connected to a charge-coupled device color camera, with identical exposure settings used for directly-matched control versus test conditions (Cell B imaging software, Olympus, Hamburg, Germany); or on an LSM510-Meta confocal microscope (Carl Zeiss, Oberkochen, Germany) using 63× oil immersion lenses and identical thresholding for control versus test samples.