Lsm5 live configuration variotwo vrgb

We detected apoptosis by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) using the ApopTag Plus Peroxidase In Situ Apoptosis Kit (Millipore, MA, USA). Kidney sections were prepared according to the procedure of the Vectastain Elite ABC kit (Vector Laboratory, CA, USA). Anti-CD3 antibody (Santa Cruz Biotechnology, CA, USA) and anti-F4/80 antibody (Abcam, Cambridge, UK) were used and revealing reaction was performed with an AEC Chromogen Kit (Immunotech, Marseille, France). All of these sections were examined in a blinded manner using light microscopy. The number of positive cells was quantified per high power field for each kidney and at least 20 fields were reviewed for each slide.
For immunofluorescence staining, sections were incubated with anti-EP4 antibody (Santa Cruz Biotechnology) and anti-aquaporin-1 (AQP-1) antibody (Millipore) overnight and then visualized with anti-rabbit Alexa Fluor-488 and -594 (Life Technologies, OR, USA). The images were captured by confocal microscopy (LSM5 Live Configuration Variotwo VRGB; Zeiss, Germany).

The PI uptake assay described by Jayathilaka et al. [52 (link)] was conducted, with some modifications, to detect alterations in the membrane permeability of A. baumannii upon treatment with Octominin. In this study, the PI uptake assay was coupled with FDA staining. In brief, the MIC (5 µg/mL) and MBC (10 µg/mL) of Octominin, 50 µg/mL of chloramphenicol (positive control), and PBS (negative control)-treated A. baumannii were incubated at 25 °C for 10 h. The cells were pelleted after centrifugation at 1500× g for 10 min and resuspended in 1 mL PBS. The bacterial cells were incubated with 50 µg/mL of PI (Sigma Aldrich, Munich, Germany) and 40 µg/mL of FDA (Sigma Aldrich, Munich, Germany) at room temperature (26 ± 2 °C) for 30 min in the dark. Excess PI and FDA were washed using PBS, and the cells were observed under a confocal laser scanning microscope (CLSM) (LSM5 Live Configuration Variotwo VRGB, Zeiss, Oberkochen, Germany). The red fluorescence was measured at excitation and emission wavelengths of 535 and 617 nm, respectively, and the green fluorescence was measured at excitation and emission wavelengths of 488 and 535 nm, respectively.