Axioimager m2 microscope

HFFs plated on glass coverslips were treated with either 50 ng/mL of Activin A or parasite-infected (MOI of 4) for 2 h and then fixed with 2% paraformaldehyde (in phosphate buffered saline (PBS) for 20′ at room temperature and then washed 3 times with ice-cold PBS. Cells were permeabilized with 0.1% Triton X-100 for 30′, blocked overnight with 3% bovine serum albumin in PBS at 4°C. Then, they were incubated with primary antibodies (Tables) for 2 h, washed 3 times in PBS and incubated with Alexa 488- or Alexa 594-conjugated secondary antibodies for 1 h. Coverslips were washed and then mounted on slides in Vectashield-containing DAPI (Vector Labs, Burlingame, CA) mounting medium. Coverslips were imaged by fluorescence microscopy and images acquired using a 100 X Plan Apo oil immersion 1.46 527 numerical aperture lens on a motorized Zeiss Axioimager M2 microscope equipped with 528 an Orca ER charge-coupled-device (CCD) camera (Hamamatsu, Bridgewater, NJ).

We prepared a frequency map from each cochlea using the Measure Line.class ImageJ plugin (available from Eaton-Peabody Laboratories Histology Resources) and the calculations were done according to Müller et al.^{24 (link)}. OHC loss was counted from DAPI/phalloidin/Myo7a/CtBP2-stained whole mounts. Lack of both the nucleus and the positively stained cell body was used as the criterion for the absence of a cell. OHC loss was counted across the cochlear duct, from the basalmost hook region to the apex. Image stack acquisition for cell counts was performed with Axio Imager.M2 microscope equipped with Apotome 2 structured illumination slider, using PlanApo ×10 objective (NA 0.45) and a z-step-size of 1.5 µm, CMOS camera (Hamamatsu ORCA Flash 4.0 V2) and Zen 2 software (all from Zeiss). Percentage of lost OHCs was counted from the total OHC population. In addition, 8-to-10-week old control and cKO mice were used to count the percentage of OHC loss at the 16, 32, and 45 kHz frequency regions. The frequency-specific cell counts were done from ×40 image stacks where the countable area included ~140 OHCs.

Specimens of Borrelia cultures or cells were examined on a Nikon Eclipse E800 microscope equipped with differential interference contrast (DIC) and epifluorescence illumination, and recorded with a Spot slider color camera. Biofilm of B. burgdorferi in the 96-well plate was visualized using the Nikon TE200 inverted microscope equipped with epifluorescence, and recorded by a monochrome camera. Zeiss AxioImager M2 microscope equipped with ORCA-R² high resolution digital camera (HAMAMATSU, Japan) was used to observe spirochetes stained by Sytox Green/Hoechst 33342 assay (see below).

To measure GFP intensity, synchronized populations of worms were anesthetized and arranged on an agarose pad. The intestinal SKN-1::GFP was assayed by confocal microscopy (X40) (Nikon A1R, Nikon Instruments Inc., Melville, NY, USA) (Fig. 4a). The quantification of intestinal SKN-1::GFP was recorded as high (≥15 GFP-positive intestinal nuclei), medium (5–15 GFP-positive intestinal nuclei), or low (≤5 GFP-positive intestinal nuclei) (Fig. 4b). ASI SKN-1::GFP was assayed by a Zeiss AxioImager M2 microscope equipped with a Hamamatsu Flash 4.0 Scientific c-mos camera and Zen2 software (×40) (Fig. 4c). The quantification of ASI SKN-1::GFP was performed with ImageJ using a sliding paraboloid algorithm for reducing the background followed by edge detection. The gst-4p::GFP intensity was measured same as ASI SKN-1::GFP but with ×5 magnification and the quantification was done by ImageJ using the whole worm signal.