Plan apo

Z-stacks images were captured using an inverted microscope (Ti-E) and a confocal laser microscope system (C2 Plus; Nikon) equipped with an objective lens (PlanApo λ 20×/0.75 NA; Nikon, Plan Apo λ 100×/1.45 NA Oil; Nikon). Cells were maintained in RPMI supplemented with 5% FBS and 10% HS and warmed in a chamber set at 37 °C (INUBTF-WSKM-B13I; Tokai Hit) during observation. Images were captured and analyzed using NIS-Elements C software. For 3D time-lapse imaging, cells were maintained in DMEM/F12 (1:1) supplemented with 10% FBS and 4.5 ng/ml NGF, and warmed in a chamber set at 37 °C during observation. EGFP and QD were excited by a laser at 488 and 561 nm, respectively. Z-stack images were captured every 15 min and were oversampled by taking 1000 nm z-steps between acquired images and were analyzed using NIS-Elements C software. For observation of Aβ₄₂ aggregation by the MSHTS system, 25 μM Aβ₄₂ in and 25 nM QDAβ, PBS containing 5% EtOH and 3% DMSO were incubated in a 1536-well plate (782096; Greiner) warmed in a chamber set at 37 °C. Z-stack images were captured every 3 min and were oversampled by taking 2500 nm z-steps between acquired images and were analyzed using NIS-Elements C software.

Drugs were added to MCF7-EGFP-α-tubulin cells in 1% FBS for 4 h after which coverslips were placed in recording media (10% FBS-DMEM, lacking phenol red and sodium bicarbonate, but supplemented with 15 mM HEPES, 3.5 g/L glucose, Oxyrase (1:50 dilution), and DL-lactate (10 mmol/L)). Cells were visualized using a 100× Nikon Plan Apo objective (N.A. 1.4, oil immersion) at 37°C on a Nikon Eclipse E800 microscope (Nikon; Tokyo, Japan) equipped with a CoolSNAP HQ2 camera (Roper Scientific GmbH, Ottobrunn, Germany). Images were taken at 4-s intervals for 2.5 min using an exposure time of 600 ms, no binning, and an 8-bit image auto scale using Metamorph software (Molecular Devices, Sunnyvale, CA) [28 (link)]. Plus ends of microtubules were tracked using Igor Pro 6.22A: Microtubule Life History Analysis Package designed by Dr. Emin Oroudjev (University of California Santa Barbara, 2010). Dynamic instability parameters were determined as described [28 (link)]. A minimum of 30 microtubules were measured from three independent experiments per condition, and reported as mean ± SEM.

Detection of B. pertussis in the nasal cavity and lung was quantified via IF and confocal imaging. The left lobe of the lung and nasal cavity were preserved and sectioned as described above. Sectioned samples underwent deparaffination and rehydration using xylene and ethanol (70 to 100%). Antigen retrieval was performed by incubating samples in citrate buffer at 98°C for 20 min. Samples were blocked using 5% bovine serum albumin (Fisher Scientific, catalog no. 159008) for 1 h and primarily labeled utilizing a polyclonal rabbit FHA antibody (a gift from Erik Hewlett) diluted in 1× PBS. Secondary labeling occurred utilizing an anti-rabbit IgG conjugated with Texas Red (Fisher Scientific, catalog no. T2767) diluted in 1× PBS. Samples were then covered in mounting media (Prolong Gold Antifade reagent with [4′,6′-diamidino-2-phenylindole], catalog no. 8961). Samples were imaged using a Nikon A1R confocal microscope. Images were analyzed on DAPI channel and at wavelength 650 nm for Texas Red acquisition. Images were acquired using a 100× oil immersion lens (100×/1.40 Nikon Plan APO). To identify any potential differences in IF between the two strains, all IF images were deidentified, and the microcolonies were manually counted blindly by four volunteers with two to three fields of view used per sample.

Golgi–Cox staining was performed using an FD Rapid GolgiStain Kit (FD Neurotechnologies) according to the manual and as previously described [45 (link)]. Following impregnation steps, the tissues were shock-frozen in iso-pentane solution precooled in dry ice and afterwards cut with cryostat in sections with 90 μm thickness and mounted in gelatin-coated glass slides. The cut tissues were allowed to dry for at least overnight, then stained by immersing them in a mixture of staining solution from the kit, dehydrated in ethanol, cleared in xylene, and mounted using Eukitt (O. Kindle) mounting media.
For analysis purposes, only impregnated neurons with diameter of at least 20 μm that were located within the laminae II to V of the spinal dorsal horn were chosen. Moreover, only secondary or tertiary dendrites that were projected in the direction of the dorsal horn were further analyzed. Images were captured using an upright microscope (Nikon NiE, Nikon) equipped with Nikon Plan Apo objective set and high resolution CCD camera (Nikon DS-Ri1, Nikon). Acquisition process was driven by NIS-Elements software 4.1 (Nikon).