Neutral buffer

PD model evaluation was performed mainly by immunohistochemistry and rota-rod test. Due to the decrease of the positive tyrosine hydroxylase (TH⁺) in the substantia nigra (SN) induced by 6-OHDA administration, the immunohistochemistry was conducted to detect TH⁺. At the beginning, the whole brains were removed and fixated in a formalin solution with 10% neutral buffer (Sigma, St. Louis, US). After completing the fixation, the area containing SN was obtained from the brains, and its dissection was performed (slice thickness: 2.5–3 μm). Then, the prepared sections were stained using the primary antibody of Rabbit anti-Tyrosine hydroxylase (MilliporeSigma, Burlington, US), and they were incubated overnight at 4 °C. The stained sections were imaged by a motorized microscope (Olympus, Tokyo, Japan), and the captured images were stored in a magnification of 400 times (ocular: × 10 and objective: × 40) of the actual size. For the comparison of the affected with the unaffected area, the detected TH⁺ was estimated by its different density by counting the stained target neurons.

UASMC were transfected with siRNA to ZIPK or ROCK1 or with negative control siRNA as described above for 30 h. The medium was replaced with fresh complete medium (containing 5% FBS) and the cells were cultured for a further 18 h. 5-bromo-2-deoxyuridine (BrDU; 10 μM) was then added to the culture medium and the cells were cultured for an additional 1 h. The cells on coverslips were fixed with 10% formalin in neutral buffer (Sigma) and BrDU was detected with Alexa Fluor 488-conjugated anti-BrDU antibody (Invitrogen).

The pancreatic tumor organoids were passaged as described previously. The immunofluorescence staining was performed as follows; The organoids were fixed in tissue fixation buffer containing formalin solution, 10% neutral buffer overnight (Sigma Chemical, USA). The following day, the fixing was stopped, and organoids were washed with 1 X PBS 3 times for 5 minutes each. Subsequently, organoids were treated with 0.2% Triton X-100 followed by blocking with 5% goat serum with 1% BSA for 1 hour. We used α SMA primary antibodies, rabbit polyclonal unconjugated at 1:100 dilution (ABclonal, USA) and Alexa fluor 594 conjugated anti-Vimentin (mouse) at 1:300 dilution for overnight in 1:5 dilution in blocking buffer. After completion of the incubation, primary antibodies were removed, and organoids were washed as described above. In addition, Alexa fluor 488 rabbit secondary antibody (ThermoFisher Scientific, USA) was used at 1:300 dilution for 1 hour to detect unconjugated αSMA. Images were captured using EVOS Microscope (ThermoFisher Scientific, USA) under dark condition.