Fluorescein isothiocyanate (fitc)

The fixed tissues were thoroughly washed in 0.01 M phosphate buffer (pH 7.4), dehydrated in graded ethanol, toluene-cleared, and embedded in paraffin. A paraffin section of testis was cut at 5 μm, mounted on slides. Fixed sections were soaked in 3% H₂O₂ and incubated for 30 min; 1% Triton X-100 for 30 min; blocked for 25 min at room temperature by the drop wise addition of 5% BSA, primary antibody (1:500) 4 °C overnight; then incubated donkey anti-rabbit IgG (1:100) secondary antibody Texas red and FITC (Invitrogen Life Technologies, Inc, CA, USA) at 37 °C for 1 h; the above steps were repeated with 0. 01 M (pH 7.2–7.4) PBS and washed twice for 5 min each wash, then VECTASHIELD mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector, Laboratories, USA) was added. A confocal laser microscope (LSM, Carl Zeiss AG, Germany) was used with the camera section on a Texas red (ΔNp73) and FITC green (Tap73) fluorescence was positive.

The nrGO-PEG was labeled by fluorescein isothiocyanate (FITC, Sigma). In brief, the solution of nrGO-PEG (approximately 0.5 mg/mL) was mixed with 0.1 mL FITC (13 mM) dissolved in DMSO and then stirred overnight at room temperature. The resulting mixtures labeled with FITC were filtrated through 30 kDa filters to remove excess unbound FITC and centrifuged at 12,000× g for 30 min to eliminate solid aggregated FITC. The obtained nrGO-PEG/FITC was re-dispersed in distilled water. The whole procedures were operated in the dark place.
A549 cells (1 × 10⁵ cells) were incubated with 100 μg/mL of nrGO-PEG/FITC and free FITC for 2 h, respectively, in the dark. After that, the cells were rinsed by phosphate buffered saline (PBS) five times. Fluorescence emission from FITC was observed using a confocal microscope (LSM 510/ConfoCor 2, Zeiss, Jena, Germany). FITC was excited at 488 nm laser with an Ar-Ion laser (reflected by a beam splitter HFT 488 nm), and fluorescence emission was recorded by a 505 to 550-nm IR band-pass filter. The uptake ratio of nrGO-PEG by A549 cells were measured by flow cytometry (FCM, FACSCantoII, Becton Drive, New Jersey, USA) using FITC labeled on nrGO-PEG, and for each FCM analysis, 10,000 events were recorded.

Osteoblasts cultivated on titanium implants for 3, 14, 21 and 28 days in complete medium with FCS, were fixed with 4% paraformaldehyde solution and permeabilized with 0.1% Triton X-100 for 20 min at room temperature. The samples were kept for 20 minutes at room temperature with 10% BSA to avoid non-specific antibody binding. Osteopontin, osteocalcine, and collagen 1A1 (all mouse anti-human primary antibodies from Santa Cruz Biotechnologies) were diluted at a ratio of 1:50 in 1% BSA and incubated overnight at 4°C with the samples. Secondary goat anti-mouse antibodies IgG1 marked with FITC (fluorescein isothiocyanate) and Texas Red (Santa Cruz Biotechnologies) were added and incubated for 45–60 minutes at 37°C. Samples were counterstained with an antifade medium containing DAPI in order to highlight the nuclei and were subsequently examined with a reversed phase epifluorescence Zeiss Axiovert D1 microscope at 488 nm for FITC, 546 nm for Texas Red and 340/360 nm for DAPI.

For primary cell cultures, 7 to 12 independent randomly pictures of every condition were taken under an Axiovent inverted microscope equipped with DAPI, FITC, and rhodamine epifluorescence filters (Zeiss, Jena, Germany). Counting was semiautomated on seven fields/condition minimum under ImageJ, using both automatic thresholding and cell counting for KI67, MAP2, and nestin and manual counting for Hoechst blue stained nuclei. Three independent experiments (> 2000 cells analyzed per condition and experiment for neurons and > 10,000 cells for neural progenitors) were quantitated.

Fluorescence in situ hybridization (FISH) was performed according to Alquéres et al. (2013 (link)), with modifications as described in Kuhl et al. (2021 (link)), using chemicals from AppliChem (Darmstadt, Germany). Briefly, the roots were desiccated in an increasing ethanol series (50%, 80%, 96%) for 3 min each. Afterward, the roots were submerged in a hybridization buffer, containing 15 pmol of the fluorescently labeled probes EUB338, specific for bacteria (Amann et al., 1990 (link); Daims et al., 1999 (link)) and labeled with fluorescein [FITC (488 nm), Thermo Scientific, Bremen, Germany], and Gam42a (Manz et al., 1992 (link)), specific for gamma-proteobacteria labeled with Cy3 (561 nm, Thermo Scientific). Hybridization was performed for 1.5 h at 46°C. Bacterial cells on the roots were investigated using confocal laser scanning microscopy (CLSM) at the Zeiss LSM 880 (Zeiss, Jena, Germany), equipped with an argon ion laser and a helium neon laser for excitation of FITC (488 nm), Cy3 (561 nm), and an unlabeled control channel (633 nm). Bacterial cells were observed with a C-Apochromat 63x/1.20 W Korr M27 water immersion objective. Micrographs were taken using Zeiss software Zen Black Edition 2.3 SP1 FP1 (Version 14.0.12.201, Zeiss).