Ab13970

To prepare for antibody staining, slides were thawed then rehydrated in PBS. In order to effectively stain myelin proteins, tissue was put through ascending, then descending ethanol dilutions (50, 70, 90, 95, 100, 95, 90, 70, 50%), followed by three washes of PBS. Tissue was then blocked with 10% normal donkey serum dissolved in PBS with 0.1% Triton X-100 for 30 min. Primary antibodies were diluted in PBS with 0.1% Triton X-100 and applied to the slides overnight at room temperature in a humid chamber. The following morning, slides were washed and incubated with donkey Dylight or Alexa Fluor secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.) for 2 h, then washed again before being coversliped using Fluoromount-G (Southern Biotech, 0100-01). Antibodies used were raised against the following antigens: CC1 (1:300, Millipore, OP80), OLIG2 (1:500, Millipore, AB9610), MYRF (1:300, N-terminus, generously provided by Dr. Michael Wegner), GFP (1:4000, Abcam, ab13970), GFAP (1:1000, Sigma, G3893), PDGFRα (1:200, R and D Systems, AF-307-NA), NF200 (1:1000, Sigma, N0142), SMI312 (1:1000, Covance, SMI-312R-100) and P0 (1:100, Aveslabs, PZO).

All animal experiments were performed according to protocols approved by the animal welfare committee of the Leiden University Medical Center and conform the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. Female Rosa26^mTmG/mTmG mice were set-up for timed matings with male Wt1^creERT2/+. The presence of a plug in the morning was denoted as embryonic day (E)0.5. At E9.5, the mother was injected with tamoxifen (2 mg) to label the pro-epicardial cells. After three days, when the epicardium has covered the heart, embryos were isolated from which the embryonic heart was dissected and cultured based on a previously published protocol (Jackson-Weaver et al., 2020 (link)). Embryonic tissue was cultured in DMEM high glucose and M199 mixed in a 1:1 ratio supplemented with 0.1% FBS and 100 U/mL penicillin and 100 mg/ml streptomycin at 37°C in 5% CO₂. After 24 h, the culture medium of the embryos carrying the Cre + genotype was supplemented with 2 ng/ml FGF with or without 200 ng/ml Activin A and incubated for another 48 h. The tissue was fixed in 4% PFA, embedded in paraffin, sectioned, and stained as described (Kruithof et al., 2020 (link)) using the following antibodies: α-GFP(Abcam, ab13970), α-Tropomyosin (Sigma-Aldrich, T9283) and α-Wt1 (Abcam, ab89901).

Frozen samples were sectioned at 18–30 µm depending on specific experiment. If needed, sections were stored at −20°C after drying 1 hr at room temperature, or processed immediately after sectioning. Primary antibodies used were: chicken anti-GFP (Abcam, 1:500, ab13970), rabbit anti-SOX9 (Sigma Aldrich, 1:1000, HPA001758), sheep anti-ERBB3 (RnD Systems, 1:500, AF4518). For detection of above-mentioned primary antibodies, we utilized 405, 488, 555 or 647-conjugated Alexa secondary antibodies produced in donkey (Invitrogen, 1:1000). Slices were mounted with 87% glycerol mounting media (Merck).

Mice received a lethal i.p. dose of pentobarbital (Fatal Plus, Vortech) followed by transcardial perfusion with 0.2M PBS (pH 7.4) and then 10% neutral-buffered formalin (Fisher Scientific). Brains were removed, postfixed, and coronally sectioned into four series of 30 μm sections using a freezing microtome (Leica). Free-floating sections were exposed to primary antibodies including chicken-GFP (1:2000, Abcam, ab13970) and mouse anti-TH (1:1000, Millipore, AB_2201528) followed by species-specific secondary antibodies conjugated to Alexa-568 (1:200, LifeTech, AB_2534017) or Alexa-488 (1:200, Jackson ImmunoResearch, 703-545-155). Sections mounted onto slides were analyzed via an Olympus BX53 fluorescence microscope with FITC and Texas Red filters. Images were collected using Cell Sens software and a Qi-Click 12 Bit cooled camera and analyzed using Photoshop (Adobe). Masks applied in Photoshop to enhance brightness and/or contrast were applied uniformly to all samples.

Immunofluorescence was performed as previously described [19 (link)]. The primary antibodies and dilutions were as follows: anti-Calretinin (Millipore, AB5054, 1:500); anti-Calbindin (Millipore, AB1778, 1:250); anti-Foxg1 (Abcam, ab18259, 1:1000); anti-GFP (Abcam, ab13970, 1:1000); anti-L1 (Millipore, MAB5272, 1:500); anti-Pax6 (BioLegend, 901,301, 1:1000); and anti-Vglut2 (Millipore, MAB5504, 1:500). The secondary antibodies used were Alexa Fluro 488 donkey anti-chicken (Jackson Lab, 703–545-155, 1:500), Alexa Fluor 488 donkey anti-rabbit (Life, A21206, 1:500), Alexa Fluor 546 donkey anti-rabbit (Life, A10040, 1:500), Alexa Fluro 647 donkey anti-rabbit (Life, A31573, 1:500), Alexa Fluor 488 donkey anti-rat (Life, A21208, 1:500), CF 633 donkey anti-rat (Sigma-Aldrich, SAB4600133, 1:500) and Alexa Fluro 647 donkey anti-mouse (Invitrogen, A21236, 1:500).