Alexa fluor 568 anti mouse igg

Mouse anti-paxillin (BD 610052); rat anti-active integrin β1 (9EG7; BD 553715); rabbit-anti-Tom20 (Santa Cruz sc-11415); mouse-anti-MYH1/2 (Santa Cruz sc-53088); rabbit anti-fibronectin (Santa Cruz sc-9068); rabbit anti-GAPDH (GeneTex GTX100118); rabbit anti-β-actin (GeneTex GTX100313); rabbit anti-integrin β1 (GeneTex GTX128839); rabbit anti-paxillin (GeneTex GTX125891); rabbit anti-pY397-FAK (GeneTex GTX129840); rabbit anti-kinesin-1 (Abcam AB167429); rabbit anti-GFP (Abcam AB290); mouse anti-α-tubulin (Sigma T5168); mouse anti-MYH2 (Thermo Fisher 14-6503-80); rabbit anti-FAK (Thermo Fisher AHO0502); Alexa Fluor 488 phalloidin (Thermo Fisher A12379); Alexa Fluor 568 phalloidin (Thermo Fisher A12380); Alexa Fluor 488-anti-rabbit IgG (Thermo Fisher A11034); Alexa Fluor 488-anti-mouse IgG (Thermo Fisher A11029); Alexa Fluor 488-anti-rat IgG (Thermo Fisher A11006); Alexa Fluor 568-anti-rabbit IgG (Thermo Fisher A11036); Alexa Fluor 568-anti-mouse IgG (Thermo Fisher A11031); DAPI (Thermo Fisher D1306); Mitotracker Red (Thermo Fisher M7512); HRP-AffiniPure mouse anti-rabbit IgG (Jackson ImmunoResearch 211-032-171); and HRP-AffiniPure goat anti-mouse IgG (Jackson ImmunoResearch 115-035-174).

Immediately after nucleofection, 100,000 fibroblasts were seeded into each well of a 24-well plate lined with a 13 mm #0 round uncoated glass coverslip. Cells were incubated for 72 h before being fixed in ice-cold acetone: methanol (1:1, v:v) and allowed to air dry. Fixed cells were washed once with PBS to rehydrate before blocking with 10% goat serum in PBS for 1 h at room temperature. The primary antibody, Anti-fibrillin-1 antibody clone 26 (Merck Millipore, Sydney, Australia), was applied at a dilution of 1:100 in 1% goat serum-PBS and incubated overnight at 4 °C. Secondary antibody; AlexaFluor568 anti-mouse IgG (Thermo Fisher Scientific, VIC, Australia) was applied, 1:400, for 1 h at room temperature, and co-stained with Hoechst 33,342 (Sigma-Aldrich, Sydney, Australia) for nuclei detection (1 mg/mL diluted, 1:125). Coverslips were mounted using ProLong™ Gold antifade mountant (Thermo Fisher Scientific, Melbourne, Australia). Fibrillin-1 was detected using a Nikon 80i microscope with NIS-Elements software (Nikon, Adelaide, Australia). The brightness and contrast of individual channel images were altered equally for each image, then merged. The merged image was cropped from the original 1280 × 1024 pixel image using Adobe Photoshop CC. A 20 µm scale bar was added using ImageJ software (NIH, Bethesda, MD, USA) [81 (link)].

Immunohistochemistry was performed as previously described [24 (link)]. For Pdf staining, brains were stained with a mouse anti-Pdf antibody (PDF C7-s, Developmental Studies Hybridoma Bank at the University of Iowa, 1:200) followed by Alexa Fluor 568 anti-mouse IgG (A11004, Thermo Fisher Scientific) as the secondary antibody (1:1,000). For GFP staining, brains were stained with a rabbit anti-GFP antibody (A11122, Thermo Fisher Scientific, 1:200), followed by Alexa Fluor 488 anti-rabbit IgG (A11008, Thermo Fisher Scientific, 1:1,000) as the secondary antibody. Fluorescence signals were observed under a confocal microscope [(LSM710, Zeiss, Germany) or (C2, Nikon, Japan)].

Kidney sections from these mice were prepared in an identical fashion. Immunostaining was performed using rabbit anti-podocalyxin (R&D Systems), rabbit anti-nephrin (a gift from Dr. Larry Holzman), and mouse anti-WT1 antibodies (Santa Cruz Biotechnology). After washing, sections were incubated with a fluorophore-linked secondary antibody (Alexa Fluor 488 anti-rabbit IgG and Alexa Fluor 568 anti-mouse IgG from Invitrogen). Slides were subsequently mounted in Aqua Poly/Mount (Polysciences Inc.) and images were acquired using AxioVision IIe microscope with a digital camera.

MDCK cells were fixed in 100% methanol in Lab-TEK chambers. Immunofluorescent staining was performed using monoclonal anti-acetylated-tubulin (1:500, Sigma) and Flag (1:500, Applied Biological Materials, Richmond, Canada) antibodies. Alexa Fluor 568 anti-mouse IgG (1:1000, Invitrogen) was used as the secondary antibody. Nuclei were stained with DAPI (4′,6′-diamidino-2-phenylindole, dihydrochloride, 1:5000, Invitrogen), and cells were examined under a Zeiss LSM7 PASCAL confocal microscope. Image processing and analysis were performed with ImageJ and Adobe Photoshop software, respectively.