Image analysis system

For PIEZO1 immunohistochemistry, deparaffinized and rehydrated slides were incubated in citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0) for 10 min at 95°C. Sections were blocked with 10% Normal Goat Serum (NGS) for 1 hour and incubated with anti-PIEZO1 antibody (1:200, rabbit polyclonal, Proteintech #15939-1-AP) overnight at 4°C. Following 30 min of wash with PBS containing 0.1% Triton-X (PBS-T) sections were incubated with goat anti-rabbit secondary antibody conjugated to Alexa Fluor 647 (1:1000, ThermoFisher Scientific) for 1 hour at room temperature. Slides were washed with PBS-T for 30 min and nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; Sigma). The slides were mounted with Fluoromount-G™ (Invitrogen #00-4958-02). The images were captured using Olympus VS120 Virtual Slide Microscope and Visiopharm Image Analysis System. Chondrocyte PIEZO1 protein expression was quantified by measuring Integrated Density of each individual chondrocyte in ImageJ. Cells with circular cell shape were defined as region of interest (ROI). 20 cells at both humeral and glenoid were selected for each sample respectively after setting the threshold. The Integrated Density was then averaged for each side and summed up to represent the entire glenohumeral joint. The intensity was normalized to control.

Soft tissues were removed from left tibiae and lumbar 1 (L1) vertebrae, fixed in 10% formalin at 4 °C for 24–48 hours on a rocker and transferred to 70% ethanol until μCT scanning. Specimens were scanned at high resolution (10.5 μm) on a VivaCT 40 μCT scanner (Scanco Medical) using 300 ms integration time, 55 kVp energy and 145 μA intensity. Then these tibiae were decalcified in 10% EDTA at 4 °C for 21 days after formalin fixation and embedded in paraffin. Sections (4 μm thick) were stained with H&E for bone volume and osteoblast (OB) analysis and with TRAP-Fast Green for osteoclast (OC) analysis using OsteoMeasure system. OB and OC numbers and surfaces per millimeter bone surface were analyzed in tibial sections with point counting using an eyepiece grid and an Olympus TH4-100 microscope. Tibial and vertebral bone volumes were analyzed in digital slides generated using an Olympus VS120 Image Analysis System and automated analysis and algorithms that we developed using a Visiopharm Image Analysis System52 (link).

Excisions were made in the small intestine, and the samples were immediately washed in physiological saline and then fixed in phosphate-buffered formalin at a 70% concentration. After being set with formalin, the samples were encased in paraffin and then sectioned longitudinally at a thickness of 5 μm. Hematoxylin and eosin were used for staining after the segment of jejunum had been deparaffinized and dehydrated (Sigma). Using an Olympus BH2 microscope, observations of the microstructures of the small intestine were carried out (DP71, Olympus, Tokyo, Japan). Using an Olympus Image Analysis System, the height of the villus and the depth of the crypt of each of the ten well-oriented villi of each mouse were measured and recorded (Olympus 6.0, Tokyo, Japan).

YM-1 and KYSE-30 cell lines were transfected as described above and collected in Immunocytochemistry fixing solution (FS) (4% paraformaldehyde and 0.05% v/v F68). Cells smear was prepared by celltrazone huro (path filter-Korea). Concisely, following peroxidase blocking specific monoclonal antibody incubation was started for 30 min at 25 centigrade degree. The used antibodies include Cadherin-E master diagnostics (Mouse anti-human Cadherin E Monoclonal Antibody/Clone HECD-1) and. Anti-beta Actin (antibody (ab8227) Abcam). After rinsing for removing excess antibody, secondary antibodies (Sigma, RE7111, US) was applied. Immunocytochemical identification was performed by the Novolink™ DAB (Polymer) (Product No: RE7230-K, RE7270-K) chromogen according to the manufactures’ protocol. Lastly, hematoxylin was applied as a counterstained. All photos were captured via a digital camera (Olympus image analysis system, Japan). Two expert pathologists who were blinded to the primary research, independently scored the immunocytochemical staining of, E-cadherin, according to the semi-quantitative immunoreactivity score. The intensity of immunostaining was scored as 0–3 (0, negative; 1, weak; 2, moderate; and 3, strong), and the percentage of immunoreactive cells was analyzed utilizing Image J software.

Five-micron formalin-fixed, paraffin-embedded tissue sections were used for hematoxylin and eosin (H&E) staining and immunofluorescence analyses. The tissues were dewaxed and rehydrated through a series of xylene and ethanol changes. For antibody staining, antigen retrieval was performed on the slides by incubating them in a steamer for 40 mins in citrate buffer (Vector Labs, Cat# H-3300-250). The slides were washed in water and then blocked using 5% goat serum in PBS for 1 hr at room temperature prior to adding primary antibodies diluted in the blocking buffer. Primary antibody (NQO1; Abcam Cat# ab196196) incubation was carried out overnight at 4° C. For immunofluorescence, the second-to-last wash included 1 µg/ml of 4’, 6’-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma, Cat# D9542-10mg) in PBS to stain cell nuclei. Immunofluorescence tissues were mounted with ProLong Gold (Life Technologies, Cat# P36934), while H&Es were mounted with Permount mounting media (Fisher Scientific, Cat# SP15500) and coverslipped for imaging. Immunofluorescence-stained slides were imaged using a CyteFinder II from Rarecyte. H&Es were imaged using an Olympus VS120 virtual slide microscope and Visiopharm image analysis system.