191 An in vitro model of fibrosis using crosslinked native extracellular matrix-derived hydrogels to modulate biomechanics without changing composition Immunofluorescence Staining The hydrogels were treated with avidin/biotin blocking kit (ThermoFisher) before being incubated with 0.5 μg/mL biotinylated wheat germ agglutinin (Vector Laboratories, Burlingame, USA) for 20 min at 37°C . Then the hydrogels were washed and permeabilized by incubating with 0.5% v/v Triton X-100 in HBSS for 10 min at RT and subsequently blocked in 2.5% v/v BSA + 0.1% Triton-X 100 in HBSS for 30 min at RT. Endogenous peroxidase activity was blocked by 30 min incubation in a 0.3% hydrogen peroxide solution. Afterwards, the hydrogels were incubated overnight with a mouse anti-human α-smooth muscle actin antibody (DAKO, Glostrup, Denmark) at 4°C. A rabbit-anti-mouse antibody conjugated with peroxidase (DAKO) and streptavidin conjugated with Alexa Fluor 555 (ThermoFisher) were used as a second step for 45 minutes at room temperature Staining for α-SMA was then developed by Opal650 tyramide (Akoya Biosciences, Marlborough MA, USA) according to the manufacturer’s instructions. After staining with 0.1 μg/mL DAPI solution (Merck), the hydrogels were mounted with Citifluor Mounting Medium (Science Services, Munich, Germany) and fluorescence microscopy was performed to acquire images. Imaging and image analysis Fluorescent images of PSR-stained LdECM and Ru-LdECM hydrogel sections were generated with Zeiss LSM 780 CLSM confocal microscope (Carl Zeiss NTS GmbH, Oberkochen, Germany), λex 561 nm / λem 566/670 nm at 40x magnification. TWOMBLI plugin for FIJI ImageJ was used to assess the number of fibres, end points, branching points, total fibre length and alignment, lacunarity, high density matrix (HDM) and curvature of the fibres as previously described (Supplementary Figure 1) [26, 32]. Fluorescent images of cell-seeded hydrogels stained for αSMA, wheat germ agglutinin and DAPI were generated with Leica SP8 confocal microscope (Leica, Wetzlar, Germany), using λex 627 nm / λem 650 nm for αSMA, λex 555 nm / λem 580 nm for wheat germ agglutinin and λex 359 nm / λem 457 nm for DAPI at 40X and 63X magnifications. Five separate images per sample (n = 5) were used to calculate the stiffness-induced changes in the expression of α-SMA, nuclei area and eccentricity (which is also described as inverse circularity (Supplementary Figure 2). Expression of α-SMA per nuclei was calculated using built-in functions for measuring area in ImageJ. CellProfiler 4.2.1 software was used to analyse the nuclei characteristics on the DAPI-stained images as previously described [33]. Circularity of the samples were calculated from the eccentricity values using the equation (4). Imaging and image analysis Fluorescent images of PSR-stained LdECM and Ru-LdECM hydrogel sections were gen with Zeiss LSM 780 CLSM confocal microscope (Carl Zeiss NTS GmbH, Oberkochen, Ger λex 561 nm / λem 566/670 nm at 40x magnification. TWOMBLI plugin for FIJI ImageJ w to assess the number of fibres, end points, branching points, total fibre length and alig lacunarity, high density matrix (HDM) and curvature of the fibres as previously de (Supplementary Figure 1) [26, 32]. Fluorescent images of cell-seeded hydrogels stai αSMA, wheat germ agglutinin and DAPI were generated with Leica SP8 confocal micr (Leica, Wetzlar, Germany), using λex 627 nm / λem 650 nm for αSMA, λex 555 nm / λem for wheat germ agglutinin and λex 359 nm / λem 457 nm for DAPI at 40X a magnifications. Five separate images per sample (n = 5) were used to calculate the st induced changes in the expression of α-SMA, nuclei area and eccentricity (which described as inverse circularity (Supplementary Figure 2). Expression of α-SMA per nu calculated using built-in functions for measuring area in ImageJ. CellProfiler 4.2.1 so was used to analyse the nuclei characteristics on the DAPI-stained images as pre described [33]. Circularity of the samples were calculated from the eccentricity value the equation (4). ( ) = 1− ( ) (4) Statistical analysis All statistical analyses were performed using GraphPad Prism v9.1.0 (GraphPad Compa Diego, USA). Data are presented as mean values with standard deviation (SD). All da (4) 8
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