Dp21 digital camera

All DAB IHC-stained slides were viewed and photographed using the Olympus BX53 bright field microscope with an Olympus DP21 digital camera (Olympus, Tokyo, Japan). The Olympus FV1200 confocal microscope (Tokyo, Japan) was used for the IF IHC-stained slides with subsequent 2D deconvolution using CellSens Dimension 1.11 software (Olympus).

The corneal irregularity was investigated in rats from each group. Briefly, reflected lines of ring-shaped light from the fiber-optic ring illuminator of a stereomicroscope (SZ51; Olympus, Japan) were lighted on the corneal surface of anesthetized rats, and the reflected lines of the light were captured with a DP21 digital camera (Olympus, Japan). Scores of corneal irregularity were graded according to the number of distorted quadrants in the reflected white ring as follows: 0, no distortion; 1, distortion in one quadrant; 2, distortion in two quadrants; 3, distortion in three quadrants; 4, distortion in all four quadrants; 5, severe distortion in which no ring could be recognized.

Corneal irregularity induced by the tear film instability was evaluated using the previously reported method [4 (link)]. All mice were anesthetized with ketamine (80 mg/kg) and xylazine (10 mg/kg). A ring-shaped light from the fiber-optic ring illuminator of a stereomicroscope (SZ51; Olympus, Tokyo, Japan) was projected on the corneal surface, and the reflected lines of light were captured with a DP21 digital camera (Olympus, Tokyo, Japan). The line shape reflected on the surface of the cornea using a circular light of the ring illuminator attached to the stereomicroscope (Olympus, Tokyo, Japan) was scored according to the following criteria: 0, perfect circle shape; 1, distortion at 1/4 of the circle; 2, distortion at 2/4 of the circle; 3, distortion at 3/4 of the circle; 4, distortion across 4/4 of a circle; and 5, severe distortion that does not recognize the shape of the circle.

All DAB IHC-stained slides were viewed, and the images were captured using an Olympus BX53 light microscope fitted with an Olympus DP21 digital camera (Tokyo, Japan). IF IHC-stained slides were viewed, and the images were captured using an Olympus FV1200 biological confocal laser-scanning microscope (Tokyo, Japan).

SGs collected from male Myd88^+/+ and Myd88^-/- mice (10 weeks of age) were immersed in periodate-lysine-paraformaldehyde-fixative for 6 h at 4°C, embedded in paraffin and serially sectioned at 5 µm of thickness. Sections were stained with hematoxylin and eosin and histologically analyzed. For immunofluorescent staining, the tissue sections were deparaffinized and immersed in distilled water. The sections were treated with 0.1% proteinase K in phosphate-buffered saline (PBS) for 5 min at room temperature and washed three times with PBS. The sections were then blocked with 1% bovine serum albumin (BSA) in PBS followed by incubation for 1 h at room temperature with rabbit anti-SLPI (secretory leukocyte peptidase inhibitor) antibody (OAPB00538; Aviva System Biology, San Diego, CA, USA). After washing three times with PBS, the sections were incubated for 30 min at room temperature with Alexa Fluor 488-conjugated anti-rabbit IgG antibody (Life Technologies, Rockville, MD, USA) and propidium iodide. After washing with PBS, the sections were sealed in the presence of Prolong Gold anti-fade reagent (Life Technologies). Optical and fluorescent images were obtained using an SZ stereomicroscope with DP21 digital camera (Olympus, Tokyo, Japan), a BX41 microscope (Olympus) and, a Biozero fluorescence microscope (KEYENCE, Osaka, Japan) and processed using Adobe Photoshop (Adobe, San Jose, CA).

Using the Olympus BX53 light microscope fitted with an Olympus DP21 digital camera, cell counting was performed using ImageJ (https://imagej.net/Welcome) on six fields of view of each DAB IHC-stained slide of the nine LMCA samples at 400x magnification. Fields of view were selected from regions that exhibited the highest density of staining. The proportion of cells within the TNs and the PTS stained positively in each field of view was calculated and the results were subjected to t-tests for related samples, using SPSS v.22 statistical package.

DAB IHC-stained slides were viewed and the images were captured using the Olympus BX53 light microscope fitted with an Olympus DP21 digital camera and processed with the CellSens 2.0 Software (Olympus, Tokyo, Japan). IF IHC-stained slides were visualized and imaged using the Olympus FV1200 biological confocal laser-scanning microscope and processed with CellSens Dimension 1.11 software using 2D deconvolution algorithm (Olympus).