Dapi d1306

Transgene expression was confirmed by visual inspection of tissue from paraformaldehyde-perfused mice. Following the completion of Ca²⁺ imaging sessions, mice were anesthetized by intraperitoneal injection of ketamine/xylazine (100 mg/kg and 10 mg/kg, respectively) and the chest cavity opened, exposing the heart. The heart was accessed by a needle and the animal was perfused at 2 ml/min with a 0.1 M phosphate-buffered (PB) solution followed by paraformaldehyde (4% by volume in PB) until the perfusate exiting an opening from the pulmonary artery ran clear. The cerebellum was removed by dissection and sliced into 80 µm sections in cold PB. When necessary, HA immunostaining was used to confirm hM4d expression. Samples were first incubated with anti-HA antibody (#ab9110, Abcam, Cambridge, UK), followed by an Alexa 633 secondary antibody (Thermo Fisher Scientific, Waltham, MA). DAPI (D1306, Thermo Fisher Scientific) counterstaining was used to identify cell locations in some cases. Images were collected on a confocal microscope (LSM 780 Axio Imager 2; Zeiss, Oberkochen, Germany) using 488 nm excitation and 493–598 nm emission for GCaMP6f, 633 nm excitation and 638–747 nm emission for Alexa 633, 405 nm excitation and 410–507 nm emission for DAPI, and 514 nm excitation and 519–620 nm emission for YFP.

The Operetta assay was performed as previously described (Schafer et al., 2017 (link)) with minor modifications: HCFs were seeded in 96-well black CellCarrier plates (PerkinElmer) at a density of 5 × 10³ cells per well. Following simulations, cells were fixed in 4% PFA (Thermo Fisher), permeabilized with 0.1% Triton X-100 (Sigma) and non-specific sites were blocked with 0.5% BSA and 0.1% Tween-20 in PBS. Cells were incubated overnight (4°C) with primary antibodies (1:500), followed by incubation with the appropriate Alexa Fluor 488 secondary antibodies (1:1,000). Cells were counterstained with 1 μg/ml DAPI (D1306, Thermo Fisher) in blocking solution. Each condition was imaged from duplicated wells and a minimum of seven fields/well using Operetta high-content imaging system 1,483 (PerkinElmer). Cells expressing ACTA2 were quantified using Harmony v3.5.2 (PerkinElmer) and the percentage of activated fibroblasts/total cell number (⍺-SMA^+ve) was determined for each field. The measurement of fluorescence intensity per area (normalized to the number of cells) of Collagen I was performed with Columbus 2.9 (PerkinElmer).

The PSC staining procedure for high-content imaging assays was performed as described previously [27 (link)]. EdU-AlexaFluor488 was incorporated using a Click-iT EdU labelling kit (C10350, ThermoFisher Scientific, Santa Clara, CA, USA) according to the manufacturer’s protocol. Primary antibodies against αSMA and collagen I (refer to Table 1 for details) were diluted in blocking solution (0.5% BSA and 0.1% Tween-20 in PBS) and incubated overnight, followed by incubation with the appropriate Alexa Fluor 488 secondary antibodies for one hour at room temperature. Cells were counterstained with 1 µg/mL DAPI (D1306, Thermo Fisher, CA, USA) in blocking solution. Two wells per condition were imaged at a minimum of 7 fields/well, using Operetta high-content imaging system 1483 (PerkinElmer). The quantification of αSMA^+ve and EdU^+ve cells was performed using Harmony software version 3.5.2 (PerkinElmer, Waltham, MA, USA). The measurement of collagen I fluorescence intensity per area (normalized to the number of cells) was performed with Columbus 2.9.0 (PerkinElmer, MA, USA).

Cells were washed with 1 X DPBS (Gibco, Thermo Fisher Scientific, USA) and fixed with 4% paraformaldehyde at room temperature for 15 min. Cells were then washed twice with 1 X PBS and permeabilised with 0.3% Triton X100 (Sigma-Aldrich, USA) in 1 X PBS for 15 min, followed by blocking with 3% bovine serum albumin in 1 X PBS at room temperature for 1 h. Incubation with primary antibodies [rabbit polyclonal MAP2 (Abcam, 32454; 1:300), mouse monoclonal Nestin (ab6320, 1:500), and mouse monoclonal alpha-synuclein (BD Biosciences, 610787; 1:200)] was performed in blocking buffer at 4°C overnight. We previously demonstrated the specificity of the alpha-synuclein antibody using alpha-synuclein knockout SH-SY5Y cells (Gao et al., 2019 (link)). The wells were then carefully washed three times with 1 X PBS and incubated for 1 h at room temperature with appropriate secondary antibodies: donkey anti-mouse Alexa Fluor 488/555 and donkey anti-rabbit Alexa Fluor 488 (Life Technologies 1:500) diluted in blocking buffer. The wells were again washed four times with 1 X PBS, and DAPI #D1306 (Thermo Fisher Scientific, USA), 300 nM, was added in the third wash to counterstain the nuclei. The wells were left in the fourth PBS wash and imaged soon after.