Tissuefaxs imaging system

mIHC was performed using an Opal 7-plex fIHC kit (PerkinElmer, Waltham, MA, USA)). Human gingival tissue Sect. (4 μm) from healthy individuals and patients with periodontitis were labeled with primary antibodies against CD68, CD86 CD206, PGRN, and TNFR2, followed by secondary antibodies. The antibodies were listed in Table 1 (in Supporting Information). Subsequently, the fluorophore-conjugated tyramide amplification system (PerkinElmer) was used for signal amplification, and DAPI was used to counterstain the nuclei. Slides were scanned using the TissueFAXS imaging system (TissueGnostics GmbH, Vienna, Austria).

Detailed materials and methods are provided in supplementary material, Supplementary materials and methods, including immunofluorescence for murine lesions with CD31 staining, immunohistochemistry for human endometrial tissues and murine lesions with Ki‐67, and active caspase‐3 staining. Images of the stained tissue were captured by the TissueFAXS imaging system (TissueGnostics, Tarzana, CA, USA) and were then processed using TissueQuest for immunofluorescence and HistoQuest for immunohistochemistry (TissueGnostics).

For CD206/IL-32/DAPI triple staining, slides were stained with rabbit anti-human CD206 (Servicebio, GB11062, China) and mouse anti-human IL-32 (BioLegend, 513501, USA) according to the manufacturer’s instructions. Sections were scanned by a TissueFAXS imaging system (Tissue Gnostics, Austria).

The samples were employed for detection and quantification of mineralisation occurred at 14 and 28 days after culturing hMSCs in the presence and absence of osteogenic factors on different surfaces. The cells were washed gently with PBS then fixed for 20 min with 4% PFA. After washing with distilled water and carefully removing the entire liquid, the samples were incubated with Alizarin Red S solution (ARS) (40 mM, pH 4.1–4.3) at room temperature for 10 min, repeatedly washed with PBS and microscopically visualised using a TissueFAXS imaging system (Tissue Gnostics, Vienna, Austria). For quantification, Alizarin red staining was extracted for 15 min at room temperature using a solution of 20% methanol and 10% acetic acid in water. Subsequently, the liquid was transferred to 96-well plates and the absorbance read at 450 nm using a Mithras LB940 Berthold spectrophotometer.

Tissue microarray for FGL1 IHC staining was obtained from the tissue bank at Urology Department of the Chinese PLA General Hospital, Beijing, China. The standard protocols were followed as previously described (23 (link)). Slides were scanned using Axio Image Z2 Microscope (Zeiss) and TissueFAXS imaging system (TissueGnostics GmbH, Austria). All images were analyzed by TissueQuest and StrataQuest software (TissueGnostics GmbH, Austria). As previously described (24 (link)), staining intensity was scored as follows: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong), whereas the staining range was scored as: 0 (0%), 1 (1%–24%), 2 (25%–49%), and 3 (50%–100%). The IHC staining score was obtained by multiplying the intensity scores with staining range. The IHC staining score ranged from 0 to 9. Scores less than two were considered as negative staining, 2–3 indicated weak staining, 4–6 was moderate staining, and >6 was strong staining. Patients with an IHC staining score ≥4 were included in the high expression group, whereas those with an IHC staining score <4 were included in the low expression group.

Tube formation assays were performed based on experiments described previously [26 (link)]. Each well of a pre-cooled 48-well plate was briefly coated with 150 μL Matrigel matrix (Corning, Tewksbury, MA, USA) and allowed to polymerize for 5 minutes at room temperature followed by 30 minutes at 37°C. Primary HPAECs or CTEPH-EC isolated from CTEPH patients (3 x 10⁴ cells per well) were seeded to the coated plate and incubated for 5h30min in EGM-2 alone or EGM-2 containing 50μM VER-155008 in a humidified incubator at 37°C with 5% of CO2. Five images were obtained from each well using TissueFAXS imaging system (TissueGnostics, Vienna, Austria).

Cells were cultured in Lab-Tek 8-chambered coverglass slides (Thermo Scientific, #155409). After treatment, cells were washed in warm PBS and fixed in 4% paraformaldehyde (PFA) plus 4% sucrose in PBS for 15 min. Cells were then incubated with PBS containing 0.3% Triton X-100 for 2 min and then blocked in 5% BSA in PBS for 1 hour at RT. After blocking, cells were incubated with Alexa Fluor 488-conjugated Phalloidin (1:500) and Alexa Fluor^® 594-conjugated Deoxyribonuclease I (1:500) diluted in 1% BSA in PBS for 1 hour at RT. Finally, cells were washed in ddH₂O and mounted with ProLong^® Gold antifade. Sections were imaged using the TissueFAXS imaging system (TissueGnostics).
Mean fluorescence intensity of phalloidin and DNAse I stainings were digitally analysed with ImageJ. Following a 300 px background subtraction, images were thresholded and the integrated density per area from the appropriate channel was calculated, using the perimeter of the cell boundary. For each data set, each replicate represents the mean of eight analysed cells. Values are expressed as arbitrary units (a.u.).