Ix70 microscope

Additionally, paraffin-embedded sections (5μm thick) were deparaffinized using xylene, rehydrated, and immunohistochemistry conducted as previously described [49 (link)]. Slides were incubated with the following primary antibodies overnight at 4°C antibodies iPLA₂β (1:100, gracious gift from Dr. Guo University of Kentucky, MMP-9 (1:500, Cell Signaling), MMP-2 (1:500, IHC World), GFP (1:500, Cell Signaling). Followed by counterstaining with hematoxylin and mounted on slides with paramount. Additionally, lung and liver sections were stained with Hematoxylin & Eosin (Surgipath). Representative images were taken under 20 to 40x magnification by Olympus IX70 microscope equipped with Olympus DP70 digital camera. Lung tumor area was analyzed using an Olympus digital camera with Olympus MicroSuit-B3 Software
Tissue mounted in Optimal Cutting Temperature compound (OCT; Tissue-Tek, Torrance, CA) was sectioned (5-μm), incubated in 4% paraformaldehyde, and blocked with normal goat serum (Vectastain ABC kit). Slides were incubated with CD31 antibody (1:200,Abcam) and counterstained with DAPI and mounted with glycerol gelatin (Sigma). Images were taken with an Olympus IX70 microscope equipped with Olympus DP70 digital camera.

Biofilms were formed by A. baumannii ATCC 17978 in flow chambers at room temperature. Images were taken at 1, 6 and 24 h (using a × 60 lens) after inoculation. A flow cell biofilm was achieved by using a flow cell chamber (ACCFL0001, Life Science Incorporated, Greensboro, NC). Briefly, overnight cultures of A. baumannii ATCC 17978 grown in MHII broth at 37 °C were diluted 100-fold with fresh MHII broth. A volume of 1 ml of dilution was injected into a flow cell chamber and allowed to settle 1 h for attachment of bacteria, followed by initiation media flow at a flow rate of 4 ml⁻¹ h⁻¹. Flow media was 10% MHII broth (control), 10% MHII broth with 7.5 μg ml⁻¹ of desferoxamine or 10% MHII broth with 7.5 μg⁻¹ ml of cahuitamycin A. Micrographs of the biofilm were acquired at 1, 6 and 24 h after the start of flow media using an Olympus IX70 microscope with a × 60 lens. Flow was still running while acquiring the micrograph.

The cells were washed with PBS and then fixed in 4% paraformaldehyde for 10 min Subsequently, the cells were washed three times with PBS and staining solution (H₂O, 1 M Tris-HCl pH 9.5, 5 M NaCl, 1 M MgCl2, NBT-BCIP (Roche) was added. The cells were incubated for 10 min in the dark at room temperature and then washed with PBS. The results were analyzed with IX70 microscope (Olympus Corporation, Tokyo, Japan).