Coolpix 995

The specimen was fixed in 10 % formalin for 24 h after marker insertion. Block-face images of the whole specimen were obtained before and after fixation using an optical/digital camera (Nikon Coolpix 995, Nikon, Japan). The block-face images served as visual reference for the registration process. The specimen was then scanned ex vivo on the CT component of a SPECT-CT scanner (Philips Precedence 16, Eindhoven, Netherlands) with careful positioning such that the orientation differences between in vivo and ex vivo CT scans of the larynx were minimised.

¹H-nuclear magnetic resonance (NMR) spectroscopy using an MR400 DD2 (Agilent Technologies, Inc., Santa Clara, CA, USA), differential scanning calorimetry (DSC) using a Q-10 (TA Instruments, Inc., New Castle, DE, USA), and POM images of LC cells using a Nikon Eclipse E600 POL (NIKON, Inc., Tokyo, Japan) equipped with a polarizer and a Nikon Coolpix 995 digital camera (NIKON, Inc., Tokyo, Japan) were employed to characterize the synthesized materials. The static contact angles of water on the polymer films were determined using a KRÜSS DSA10 (KRÜSS Scientific Instruments Inc., Hamburg, Germany) contact angle analyzer equipped with drop shape analysis software (KRÜSS Scientific Instruments Inc., Hamburg, Germany). The contact angles for each sample were measured more than four times on three independently prepared films, and the average values were used. UV stability tests were conducted on the LC cells using a VL-6.LC lamp (λ_max = 365 nm, Vilber Lourmat, Paris, France) to investigate the effects of a severe environment. The exposure dose of UV irradiation on the LC cells was measured with a UV detector using a GT-513 (Giltron, Seoul, Korea).

After harvesting the nerve cross-sections, they were stored in 4% formalin (ROTI^® Histofix 4%, Carl Roth GmbH, Karlsruhe, Germany). After washing in phosphate buffered saline (PBS) overnight at 4 °C, the nerve samples were dehydrated in increasing sucrose/distilled water solutions (10%, 20%, and 40%). The probes were embedded using Tissue-Tek^® O.C.T.^™ (Sakura Finetek Europe B.V., Zoeterwoude, The Netherlands) and shock-frozen at −80 °C in 2-methyl-butane. 15 µm thick cryostat-sections were obtained and mounted on SuperFrost Ultra Plus^© slides (Thermo Fisher Scientific/Menzel-Gläser, Braunschweig, Germany). The slides were dried at room temperature for 30–60 min and subsequently stored for 30–60 min in distilled water. After washing the slides in 50% ethanol for 1–3 min they were stained with Sudan black B staining solution for 5 min and subsequently washed in distilled water. The slides were covered using Kaiser’s glycerol gelatine (Carl Roth GmbH, Germany).
The slides were analyzed using a light microscope (Nikon Eclipse 80i, Nikon Corp., Tokyo, Japan) and photographed with the corresponding camera (Nikon Coolpix 995, Nikon Corp., Tokyo, Japan). The number of axons was determined by the ImageJ counting tool (ImageJ version 1.53a, Wayne Rasband, National Institutes of Health, Bethesda, MD, USA) (see Figure 1).

Twenty-four hours after the final challenge, to harvest the lungs, mice were euthanized by an intraperitoneal injection of a lethal dose of 10% chloral hydrate (0.3 mL/100 g, i.p). After fixing in formaldehyde for 24 hours, lungs were dissected and paraffin-embedded. The lung tissue was cut into thin slices four microns thick and then stained with hematoxylin and eosin (H&E; Sigma-Aldrich). At least five bronchi were selected from each mouse based on size (150–350 mm in diameter) for analysis. In order to reduce evaluator bias, the degree of airway inflammatory cell infiltration was scored in a single-blind fashion. Lung lesions were scored semi-quantitatively using a measurement tool as previously described^{62 (link)}. Images were captured under a Nikon Eclipse E200 microscope connected to a Nikon Coolpix 995 camera (Nikon, Tokyo, Japan). The severity of inflammation was evaluated by assigning a value of 0 point for normal; 1 point for few cells; 2 points for a ring of inflammatory cells 1 cell layer deep; 3 points for a ring of inflammatory cells 2 to 4 cells deep; 4 points for a ring of inflammatory cells of >4 cells deep.