Plan apo lens

Fluorescence images were acquired using Volocity software (PerkinElmer Life Sciences) with a Nikon Eclipse T.I confocal microscope equipped with a ×40 Plan Apo lens (Nikon). All images were captured with the same exposure and laser intensity at ×40 magnification. Images were processed using ImageJ (21 ) and JASC Paint Shop Pro V 9.0 (JASC Software Inc.). DAPI staining was used to identify the epidermal/dermal junction.

According to previously described protocol [18 (link)], cells were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 30 min and then permeabilized with methanol for 10 min at room temperature. After washing with PBS, the fixed cells were incubated with blocking buffer (1% bovine serum albumin (BSA) and 0.1% Triton X-100 in PBS) for 3 h at 4 °C followed by incubation with a primary antibody (1:300 dilution in blocking buffer) overnight at 4 °C. Methanol and Triton X-100 were not used for the ANXA2 externalization assay. The cells were then incubated with a fluorescein isothiocyanate (FITC)- or Cy3- conjugated secondary antibody (1:500 dilution in blocking buffer, Chemicon International Inc., Temecula, CA, USA). The nuclei were visualized with Hoechst 33342 (Molecular Probes, Eugene, OR, USA). Images were captured with a BZ-8000 microscope (Keyence, Osaka, Japan) with a 20× Plan APO lens (Nikon, Tokyo, Japan) or with a LSM 510 META confocal laser microscope with a 40× Plan-Neofluar lens or a 63× Plan-Apochromat lens (Carl Zeiss, Oberkochen, Germany).

Kc167 cells were challenged with L. pneumophila GFP⁺ cells and treated with 10 µg/ml of erythromycin at 19 h after challenge. Infections were allowed to proceed for an additional 11 or 21 h, at which point, the cells were fixed in 3.7% paraformaldehyde in 1× PBS and stained with 1 µg/ml Hoechst stain. Images were taken using a Molecular Devices ImageXpress automated microscope with a Nikon 20× Plan Apo lens for 2 sites per well using the DAPI (4′,6-diamidino-2-phenylindole) and FITC (fluorescein isothiocyanate) optimized filter sets. Downloaded images were subjected to an automated MetaXpress script that counts for the total area of cell nuclei (based on Hoechst staining) and bacterial replication (based on total GFP signal).

Mice peripheral tissues and specific brain regions were fixed by vascular perfusion with 30 mL of 4 % paraformaldehyde (PFA) after perfusion with PBS for about 20 min. Desired organs were isolated and washed with PBS then processed using a rapid microwave automatic histo-processor using a standard protocol and embedded into paraffin wax. Subsequent immunohistochemistry assays were then performed per standard protocols previously described [15 (link)]. Briefly, sectioned tissues were deparrafinized then permeabilized with methanol. The sections were then blocked with 3 % BSA for 1 h at room temperature and later incubated with primary antibody (1:300 mouse monoclonal IgG 2 F11; Enzo Life Sciences Int., PA, USA and AntiCT)) at 4 °C overnight, washed with PBST then with secondary antibody conjugated to AlexFluor (1:600) for 2 h at room temperature. Counterstaining with Hoechst 33342 (1:10,000) was effected. The slides were then mounted by pristine mount and left to dry overnight at 4 °C. Images were collected by BZ-8000 microscope (KEYENCE, Osaka, Japan) with a × 20 Plan APO lens (Nikon, Tokyo, Japan).