Cellsens standard imaging software

The transfected trophoblast cells were cultured in 6-well plates at 1 × 10⁶ cells per well. After complete adherence of cells to the bottom, a 2-mm cell scraper was used to scratch in the middle of each well. The medium and floating cells were washed with PBS. The cell culture was continued in renewed medium at 37°C with 4% CO₂ for 48 h. The width of the scratches at the 0 and 48 h was observed under the microscope (IX51, Olympus) and analyzed using the cellSens standard imaging software (Olympus). The migration rate was cells was measured as follow: rate = (width at 0 h -width at 48 h)/width at 0 h [27 (link)].

The frozen sections were incubated for the nicotinamide adenine dinucleotide-tetrazolium
reductase (NADH-TR) assay. NADH-TR is the mitochondrial enzyme NADH-dehydrogenase which
transfers electrons to the colorless, soluble tetrazolium salt and converts it into an
insoluble formazan compound. In this reaction, the intermyofibrillar matrix is stained and
fiber types (I and II) can be distinguished. Classification of the cross-sectional fiber
types was performed according to the staining intensities of NADH-TR. Dark fibers with
NADH-TR were identified as type I fibers and pale/light fibers with NADH-TR were
identified as type IIb fibers. Type IIa fibers were also observed with an intermediate
level of intensity with NADH-TR but were not estimated in this study. Cross-sectional
areas (shown in μm²) of the soleus (mainly composed of type I fiber) and the
type I and type IIb fibers in the EDL were measured for 50 fibers/animals and compared
with those of SD rats. For morphometric analysis, tissue images were obtained at ×400
using the cellSens Standard imaging software (Olympus, Tokyo, Japan).

Immunohistochemical staining was performed on TMA and formalin‐fixed, paraffin‐embedded tissues from PC‐3 cell line xenografts. Before staining, sections were dried for 1 hour at 60℃ and deparaffinized in xylene for 30 minutes, after which they were rehydrated by serial incubations in alcohol. Heat‐induced antigen retrieval was performed in citrate buffer at pH 6.0. Sections were subsequently blocked with goat serum and incubated with primary antibody [anti‐β₂‐AR rabbit monoclonal antibody (ab182136; Abcam), anti‐Ki‐67 rabbit monoclonal antibody (9027; Cell Signaling), anti‐Shh rabbit monoclonal antibody (ab53281; Abcam), anti‐Gli1 rabbit polyclonal antibody (bs‐1206R; Bioss, China) or anti‐Ptch1 rabbit polyclonal antibody (ab53715; Abcam) overnight at 4°C (details were shown in Table 1), followed by Histostain‐SP (Streptavidin‐Peroxidase) kit reagent (SP0023; Beijing Biosynthesis Biotec, China), according to the manufacturer's instructions. The diaminobenzidine chromogen (ZSGB Biotec, China) was used for coloration. Slides were finally counterstained with hematoxylin. Negative controls were obtained after the omission of the primary antibody. Stained sections were examined and photographed on a bright‐field microscope (BX53; Olympus) connected to cellSens standard imaging software (Olympus).

Only two samples from Blyth's hawk-eagles (BHE; Spizaetus alboniger, KU549 and KU589) were prepared using Giemsa-stained blood smears. Blood smears were prepared immediately after blood collection and then allowed to dry using an electric fan. Staining was performed as previously reported (Valkiūnas et al., 2008 (link)). Thereafter, images were obtained using a BX53 light microscope equipped with a DP73 digital camera and cellSens Standard imaging software (Olympus, Tokyo, Japan). Morphological description and morphometric measurements were not performed because parasite morphology might be influenced by EDTA (Pornpanom et al., 2019a (link)).