Rabbit anti desmin

The primary antibodies used in this study were rabbit anti-desmin (1∶50, Cell Signaling), rat anti-Sca-1 (1∶40, Sigma-Aldrich), mouse anti-S100 (1∶40, Invitrogen). Cultured mouse MDSCs, SCs and tMDSCs were fixed and stained according to standard procedures [14] (link).

Brain sections were blocked with 0.5% TritonX-100 and 10% donkey serum in PBS for 1 h at room temperature, after which the sections were incubated with primary antibodies overnight at 4°C. Primary antibodies included rat anti-CD31 (antiplatelet endothelial cell adhesion molecule-1, labels blood vessels) (Xu et al., 2017 (link)) (BD Pharmingen, 553369), rabbit anti-desmin (Cell Signaling, #5332), and rabbit anti-collagen IV (Bio-Rad, 2150-1470). The slides were washed and then incubated with Alexa Fluor 488-conjugated species-appropriate secondary antibody (Vector Labs, DI-1488) and Cy3-conjugated streptavidin (Invitrogen, A10522) for 1 h at room temperature in the dark. Then, the slides were stained with 4′,6-diamidino-2-phenylindole for 5 min in the dark, and the brain slices were washed with PBS, then patched and mounted.

Mouse embryo on E16 was fixed with 4% PFA in phosphate buffer overnight, and dehydrated and stored in methanol on − 30 °C until use. The specimen was rehydrated and stained with 1% tannic acid in PBS overnight, pre-embedded with white-agarose, dehydrated again, and embedded in paraffin. For immunostaining, paraffin sections were dewaxed, rehydrated and autoclaved at 120 °C for 15 min in 10 mM citric acid pH6.0 buffer. The sections were blocked with 5% normal donkey serum, and incubated with rabbit anti-desmin (1:100, Cell Signaling Technology #4024S, Danvers, MA, USA), rabbit anti-cyclin D1 (cl:SP4, 1:100, Thermo Fisher Scientific K.K., #RM-9104-SO, Tokyo, Japan), and goat anti-Pax3/7 (1:200, Santa Cruz Biotechnology #sc-7748, Dallas, TX, USA)^{35 (link)} for overnight at 4 °C, and followed by AlexaFluor488-donkey anti-rabbit IgG antibody (1:500, Jackson Immunoresearch, West Grove, PA, USA), Rhodamine-RedX-donkey anti-goat antibody (1:500, Jackson Immunoresearch), and DAPI (1:200 of saturated solution) for 1 h at room temperature. All micrographs were obtained using Nikon AZ-100 fluorescent microscope equipped with a CCD camera (SPOT RT3, Diagnostic Instruments, Sterling Heights, MI, USA) or a digital camera (Sony a7RII).