Das microscope

Thionine staining was used to quantify pyramidal neuron density in the hippocampus CA1 and CA3. Slides were transported in containers filled with 0.01 M phosphate-buffered saline (PBS; pH = 7.4), then soaked in dH₂O (30 s), thionine (10 min), 50, 70, 95, and 2 × 100% ethanol (2 min each), 2 × Citrisolv (2 min each; Decon Labs, Inc., United States). Slides were covered with Permount Mounting Medium, were cover-slipped, and were left to seal overnight. For each subject, six histologically representative pictures of the CA1 and CA3 hippocampal subregions were obtained using a Leica DAS microscope with an attached SONY digital camera and were recorded via Norton Eclipse 6.0 software (Empix Imaging, Mississauga, ON, Canada). Pyramidal neuron density within 1 mm linear length was quantified with ImageJ software (National Institutes of Health, United States) by two blinded examiners with achievement of inter-rater reliability. Intact neurons were defined as having a clear nuclear area enclosing a nucleolus and a cytoplasm contained within a rounded cell body.

After exposure of formalin-fixed HCT-8 cell monolayers to viable or dead S. Enteritidis (1 × 10⁸ cells/ml) for 30-min, the wells of the chambered slides (Fisher Scientific) were washed with PBS to remove unattached bacterial cells (as above). After immunoprobing with mAb-2F11, the monolayers were washed and probed with Alexa Fluor 488 conjugated anti-mouse antibody for 1.5 h at room temperature in the dark, followed by three PBS wash. Note, antibody concentrations used were the same as above. The monolayers were counterstained with DAPI (500 ng/mL; Cell-Signaling) for nuclear staining and the slides were mounted using an antifade reagent (Cell-Signaling). Images were acquired using the Nikon A1R confocal microscope with a Plano AP VC oil immersion objective (Drolia et al., 2018 (link)) and were processed with the Nikon Elements software at the Purdue Bindley Bioscience Imaging Facility.
For Giemsa staining, the formalin-fixed HCT-8 cell monolayers were exposed to viable or dead S. Enteritidis cells as above, air-dried, and immersed in Giemsa staining solution for 20 min. Giemsa staining solution was prepared using a 20-fold dilution of the KaryoMAX Giemsa staining solution (Thermo-Fisher) in deionized water. The slides were examined under a Leica DAS Microscope at the magnification of 1,000×.