Dm6b fluorescent microscope

After 4 weeks, the animals had been sacrificed, and their spinal cords were extracted and fixed with a solution of 4% paraformaldehyde and 30% sucrose. A 3-mm length of spinal cord tissue (center of the epicenter of the injured lesion) was cut into 35-μm-thick sections by an instrument (RM2135, Leica) and processed for immunohistochemistry, as described in detail [22 (link)]. The primary antibodies were rabbit anti-GFAP for astroglia (1:1000) and mouse anti-Map-2 for neurons (APC; 1:500) (Calbiochem, Germany). The secondary antibodies were Alexa Fluor 488 (green, 1:1000) (Molecular Probes, Germany) and Cy5 (red, 1:500) (Dianova, Germany). The sections were observed and photographed using a DM-6B fluorescent microscope (Leica, Germany) connected to a computer screen. The images were analyzed by ImageJ software. For the analysis of the total cavity volume in the injured lesion, the spinal cord was cut into 20-μm-thick cross-sections 4 weeks after the SCI. With hematoxylin-eosin staining, an investigator blindly calculated the volume of the cavity (details of this method were described as previous studies) [40 (link), 41 (link)].

Entrapment of E. coli cells on a peptide-modified ZnO surface was investigated. To fabricate the ZnO surface, ZnO particles (average diameter 25 nm) in milliQ (1 mg mL⁻¹) were added to a silicon rubber-encircled hole (diameter 3 mm) attached on a glass slide. By drying up the aqueous solution, a white layer was observed indicating that a ZnO surface was fabricated onto the glass slide. After washing the surface by PBS two times, a bi-functional peptide (GRHIFWRRGGGHKVAPR, 100 μM) was added and incubated for 2 h at 25 °C. Then, the surface was washed with PBS two times. Prior to the binding assay, E. coli cells were stained with SYTO®9 (150 nM) for 30 min and washed twice with PBS. The stained cells (1.0 × 10⁵ cells, 10 μL) were spotted onto the peptide-modified ZnO surface and incubated for 2 h. The incubation time was shorter during the screening process to eliminate non-specific binding to ZnO surface. Following five washes with PBS, the binding of peptide-modified ZnO and E. coli was observed using a DM6B fluorescent microscope (Leica). Bound cells were counted manually from five randomly selected areas on the images. As a negative control, ZnO particles without peptide modification were evaluated for this assay.

Embryos were dechorionated with bleach and crosslinked with 4% formaldehyde in PEM buffer (0.5 M PIPES, 5 mM MgCl₂, 5 mM EGTA, pH 6.9) in 2 ml heptane, while vortexing at medium speed for 20 min. Embryos were devitellinized in methanol/heptane and kept at −20C until needed. Embryos were immunostained as in Rothwell and Sullivan (2007) (link) using anti-H3K14ac (1:200; rabbit, 07353, Millipore), anti-HA (1:200; rat, 11867423001, Roche), anti-FLAG (1:200; rabbit, F7425, Sigma-Aldrich), anti-Drosophila Chiffon-C (1:200; rabbit), Alexa Fluor 488- and Alexa Fluor 568- conjugated secondary antibody (1:400; goat, Thermo Fisher Scientific). Cell nuclei were stained using 1 µg/ml DAPI. Embryos were staged according to the number of nuclei stained by DAPI. Images were taken using a Leica DM6B fluorescent microscope equipped with CTR6-LED and DFC450 digital camera. Acetylation levels were determined as average sum intensity values for fluorescence comparing GFP (mutant embryo) with the non-GFP embryo (WT homozygous sibling) using ImageJ software. Multiple sections were examined for each embryo (n≥3), and single optical sections are shown for each representative image.

Cells were grown on autoclaved coverslips and supplemented with 2 μg/ml Cytochalasin B prior to infection with Helicobacter pylori (P12 wild-type, P12ΔRfaE) for 12 h. Cells were washed three times with 1× PBS and then fixed with 4% paraformaldehyde for 15 min at RT. After washing three times with 1× PBS cells were stained with 1 μg/ml DAPI diluted in distilled water for 5 min at RT. Coverslips were mounted with Fluoromount-G mounting medium (Invitrogen, 00-4958-02). Images were acquired with a Leica DM6B fluorescent microscope. In each experiment micronuclei of at least 100 binucleated cells were counted per condition.