Alexa fluor 594 donkey anti goat igg h l

Skin sections (50 µm thick) were cut and transferred to a neutral PBS buffer in 48-well plates. Target retrieval (in 10 mmol/l sodium citrate buffer for 20 min at 80°C) and blocking (in 1% [wt/vol.] BSA, 0.3% [vol./vol.] Triton X-100 in PBS solution for 1 h) were performed before immunolabelling.
For the primary antibodies, SCs were labelled by rabbit anti-S100 calcium-binding protein B (S100B; the antibody is ready to use with no dilution; DAKO, Denmark) and goat anti-transcription factor SOX10 antibodies (1:100 in S100B antibody solution [ready to use], R&D system, UK), and mouse anti-PGP9.5 antibody (1:1000 in S100B antibody solution [ready to use]; Bio-Rad, USA) was used to label peripheral nerves. Skin sections were incubated with the three primary antibodies in blocking buffer over two nights at 4°C, followed by a 1.5 h incubation in secondary antibody solution. The secondary antibodies were used at a concentration of 1:650 in blocking buffer: Alexa Fluor 488 donkey anti-mouse IgG (H+L) (Abcam, UK); Alexa Fluor 647 donkey anti-rabbit IgG (H+L) (Invitrogen, USA); and Alexa Fluor 594 donkey anti-goat IgG (H+L) (Invitrogen, Waltham, MA, USA) at room temperature. Lastly, DAPI (1:5000 in PBS buffer; Sigma, St Louis, MI, USA) nucleus counterstaining was applied to the skin sections before mounting.

Retinal organoids derived from RPiPSCs or NiPSCs were washed with PBS and rapidly frozen in an embedding medium (Sakura Finetek) before sectioning. The sections (10 μm) or RPE cells were thawed, air-dried, and fixed in 4% paraformaldehyde at room temperature for 15 min. Then, they were incubated with blocking buffer (3% BSA diluted in PBS) for 1 h, and primary antibodies for 2 h at room temperature. Next, they were incubated with a secondary fluorescent antibody (goat anti-mouse IgG H&L Alexa Fluor 488, Thermo fisher; goat anti-rabbit IgG H&L Alexa Fluor 594, Thermo fisher; donkey anti-goat IgG H&L Alexa Fluor 594, Thermo fisher) for 1 h at room temperature in the dark. The nuclei were stained with DAPI for 5 min. The sections were washed with PBS between these incubations. Finally, the sections were mounted with fluorescence mounting medium, and immunofluorescent images were captured with a fluorescence microscope (Leica). Detailed information of antibodies used are listed in Supplementary Table S2.

For immunofluorescence staining of subcutaneous tumor, after deparaffinization, hydration, antigen retrieval, and serum blocking, tumor sections were incubated with primary antibodies diluted in PBS overnight at 4 °C. Alexa-conjugated secondary antibodies were used for fluorescent. Finally, slides were mounted via mounting medium with DAPI (Abcam, ab104139). The primary antibodies included ACC1 (1:100, Proteintech, 21923-1-AP), anti-ATP citrate lyase (1:100, Abcam, ab40793), Donkey anti-rabbit IgG H&L (Alexa Fluor 488) (1:200, Abcam, ab150073), Donkey anti-goat IgG H&L (Alexa Fluor 594) (1:200, Thermo Fisher Scientific, A-11058).