Alexa fluor 555 goat anti rabbit antibody

Glioblastoma stem-like cell tumorspheres or dissociated single cells were plated on Matrigel-coated coverslips, fixed in 4% paraformaldehyde for 10 min and blocked with 10% BSA/0.05% Tween for 1 h at room temperature. Primary Nilo1 monoclonal antibody was generated by the fusion of hamster B cells and the mouse myeloma X63Ag8 (13 (link)) and purified in CNB-CSIC (Madrid, Spain). Cells were incubated with Nilo1 1:100 overnight at 4°C, followed by 1:200 FITC-conjugated anti-hamster secondary antibody from BD. F-actin was stained with Phalloidin-iFluor 647 (1:40, 1 h at room temperature, Abcam) and nuclei with DAPI (1:5000, 10 min at room temperature, Sigma). For stem-like and differentiation markers we used anti-GFAP (1:500, Dako) and anti-OLIG2 (1:500, Millipore), followed by Alexa fluor 555 goat anti-rabbit antibody (1:1000) from Invitrogen. To estimate cell viability within the tumorspheres, 5 μg/ml 7-AAD (BioLegend) was used. Coverslips were mounted with Fluorsave (Calbiochem). The images were taken using the glycerol ACS APO 20x NA0.60 immersion objective of a confocal fluorescence microscope (SPE, Leica-Microsystems) and analyzed using FIJI software.

Hepatocytes (0.2 × 10⁶ cells) alone or with MSC (0.2 × 10⁶ cells, ratio 1 : 1) were seeded on 12 mm coverslips in a 24-well plate in 1 mL of complete F12 medium. After 3 days, cells were washed with phosphate-buffered saline (PBS) and fixed with a 10% formalin solution (Sigma-Aldrich, Buchs, Switzerland) for 12 min. Cells were then permeabilized with Triton X-100 0.1% diluted in PBS for 15 min, and epitopes were blocked using 0.5% bovine serum albumin (BSA) for 30 min. Hepatocytes were stained with an anti-pig albumin antibody (Abcam) diluted to 1/200 and a secondary Alexa Fluor 555 goat anti-rabbit antibody (Life Technologies) diluted to 1/500. MSC were stained with a mouse anti-human vimentin antibody diluted to 1 : 50 (Dako, Glostrup, Denmark) and a secondary Alexa Fluor 488 goat anti-mouse antibody (Life Technologies). For 5-ethynyl-2′-deoxyuridine (Edu) staining, the Click-iT EdU Alexa Fluor 488 Imaging Kit (Thermo Fisher) was used following the manufacturer's instructions. Coverslips were mounted using VECTASHIELD mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Cambridgeshire, UK). Images were acquired using a fluorescence microscope and LAS V4.5 software (Leica Microsystems).

Slides were prepared with SV40 MES13 cells stimulated by IS for 48 h, followed by incubation with fixation buffer (00-8222-49, eBioscience, San Diego, CA) for 30 min. Slides were incubated overnight at 4 °C with primary antibodies [anti-(P)RR (homemade) or normal rabbit serum (as a control); 1:5000] in protein block serum-free solution (X0909, Dako, Glostrup, Denmark). Antiserum against (P)RR was raised in a rabbit by injecting a human (P)RR fragment corresponding to amino acids 224–237 conjugated to BSA^{31 (link),32 (link)}. After washing, slides were incubated with Alexa Fluor 555 goat anti-rabbit antibody (A-21428, Life technologies; 1:1000) for 30 min in protein block serum-free solution. Then, slides were embedded in DAPI-Fluoromount-G (SouthernBiotech, Birmingham, AL) and observed by C2Si confocal microscopy (Nikon, Tokyo, Japan).

The bEnd.3 cells were seeded in confocal dish in a total volume of 2 ml complete medium and were stimulated with 0.1 μg/ml VP1 for 24 h. Then, bEnd.3 cells were washed with PBS, fixed with 4% paraformaldehyde for 30 min, blocked with 5% goat serum for 1 h, and incubated with primary antibodies against vimentin overnight at 4 °C. After incubation of the primary antibodies, bEnd.3 cells were stained with Alexa Fluor® 555 goat-anti-rabbit antibody (1:500, Life technologies, USA) in the dark for 2 h. At last, 4′6-diamidino-2-phenylindole (DAPI, 1:2000) was added for 20 min. Images were captured by ZEISS LSM710 confocal fluorescence microscope.

Neutrophils (1 × 10⁶) were seeded on a sterile round glass cover slip that was placed in a 24-well cell culture plate. As described above in the flow cytometric assay for NETs, 100 μM PMA or 1 μg/ml VP1 was added. After 4 h of incubation, the glass cover slips with the attached cells were carefully removed from a 24-well culture plate and fixed with ice-cold acetone. Then, samples were blocked with 5% goat sera and stained overnight with a rabbit polyclonal antibody to histone 3 (citrulline R2 + R8 + R17) (1:300, ab5103, Abcam) and with a mouse anti-MPO antibody (1:300, ab90810, Abcam). The samples were washed in PBST and further stained with an Alexa Fluor® 555 goat anti-rabbit antibody (1:500, Life Technologies, USA) and an Alexa Fluor® 647 goat anti-mouse antibody (1:1000, Life Technologies, USA). Nuclei in the samples were stained with 4′6-diamidino-2-phenylindole. Images were captured by Zeiss LSM7 confocal fluorescence microscopes using the appropriate lenses and filters.
For experiments in vivo, the left lower lung of mice that were or were not treated with VP1 was carefully harvested, frozen in Tissue-Tek OCT media and prepared into 7-μm-thick slices. The fixation, blocking, antibody incubation, and image acquisition of these sections were performed as described at the cell level.

Coverslips with fixed samples were first washed once with PBS. Following this step, neutrophil membranes were permeabilized using 0.5% Tween 20 for 15 min at RT. Subsequently, a blocking step was performed with 1% bovine serum albumin (BSA) for 1 h at RT. Incubation with primary rabbit polyclonal antibodies raised against whole P. gingivalis cells or whole A. actinomycetemcomitans was performed for 1 h at RT, which was followed by two washes with PBS. Subsequently, incubation with either Alexa Fluor 488, Alexa Fluor 647, or Alexa Fluor 555 goat anti-rabbit antibodies (Invitrogen) was performed for 30 min at RT in the dark. Neutrophil nuclei were stained at this step with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). In some cases, actin was visualized using tetramethyl-rhodamine B isothiocyanate-phalloidin (TRITC-phalloidin; Sigma-Aldrich). After washing two times with PBS, the coverslips were mounted on polylysine slides (Thermo Fisher Scientific) using Mowiol 4-80 as the mounting medium (Sigma-Aldrich). All washing steps and incubations were done carefully to avoid detachment of cells, especially neutrophils, from the coverslips. The mounting medium was dried by overnight incubation at RT in the dark.