Axioscope 5 microscope

Five- to 10-mm membrane fragments were dissected under a stereomicroscope and fixed in Davidson’s solution for 24 h before being processed using standard techniques for embedding in paraffin [33 (link), 34 (link)]. Two 5-μm serial sections were prepared [32 (link)] using a Microm HM 315 microtome (Microm, Walldorf, Germany), deparaffinized and stained on glass slides with haematoxylin–eosin [35 ].
The membrane fragments on the prepared slides were visualized with an Axioscope 5 microscope (Zeiss, Germany) and photographed with an Axiocam 506 camera system (Zeiss) for comparative histology. ZEN (blue edition) version 3.4 software (Zeiss, Germany) was used to measure the thickness of the membranes. A total of six samples were used for each membrane type [36 (link)]. The average thickness of the membranes was determined by measuring four sites in each section. Each site was continuous for at least 100 μm in length and had intact bedding [37 (link)].

The bacteria (S. agalactiae and A. hydrophila) were prepared as mentioned above and incubated with rGST-On-CRP, GST-tag, CaCl₂ or EDTA with the final concentration of bacteria at 10⁷ CFU/mL, rGST-On-CRP and GST-tag at 2 μM, CaCl₂ at 10 mM and EDTA at 20 mM. Then the solutions were incubated for 0.5 h at 25 °C, observed and photographed using a ZEISS Axioscope 5 microscope (Zeiss, Jena, Germany).

A previously established method (63 (link)) was modified to assess adherence of NTHi to a vitronectin-coated glass surface by microscopy. A solution of vitronectin (Recombinant human VTN, Gibco A14700) was prepared in 1× DPBS at the concentration of 2 μg/mL. Then, 10 μL was added as a drop onto a glass slide and allowed to dry for 30 min at room temperature. Similarly, a glass slide was coated with 1% BSA as a negative control. The slides were washed three times by dipping for 2 s in 1× DPBS to remove excess protein. Bacterial inoculum was prepared from log phase cultures of NTHi in 1× DPBS at a density of 1 × 10⁸ CFU/mL. The coated glass slides were submerged in 10 mL of bacterial inoculum in sterile petri dishes and incubated at 37°C, and 5% CO₂ for 1 h at 25 rpm. After incubation, the slides were washed three times in 1× DPBS to remove any unbound bacteria. The slides were air-dried, heat-fixed by passing over a flame, and stained with methylene blue (Epredia Shandon Kwik-Diff Stains) for 60 s. Excess stain was removed by washing gently with water. The slides were air-dried and imaged using the 20× objective of a Zeiss Axioscope 5 microscope. The images were processed using ZEN 3.0 software, and the area occupied by NTHi in each field of view was determined with ImageJ software. The experiment was carried out three times with at least five fields imaged for each sample.

Immediately following euthanasia, the optic nerve was removed and fixed in 2% glutaraldehyde and 2% paraformaldehyde. The nerves were then post-fixed in osmium tetraoxide and embedded into Eponate 12 resin. 1 μm thick sections of the optic nerve were taken perpendicular to the length of the nerve. Sections were then stained using 1% p-phenylenediamine (PPD) and images were taken at 100× magnification using a Zeiss Axioscope 5 microscope.