Sox2 rabbit

Whole mount immunohistochemistry was performed on previously fixed tissue^{43 (link)}. Ears were washed in phosphate buffered saline (PBS), then washed five times five minutes in PBS/0.05% Tween20 followed by blocking for one hour in 5% normal donkey serum, 1% bovine serum albumin, and 0.5% TritonX-100 in PBS. The tissue was incubated in primary antibodies, diluted in blocking buffer, at 4°C for three nights. The following primary antibodies were used: MYO6 Rabbit (Sigma; 1:1000), MYOSIN7A Mouse (DSHB; 1:200), MYSOINVIIA Rabbit (Proteus Biosciences, Inc.; 1:500), Neurofilament 200 HC Chicken (Aves; 1:200), PROX1 Goat (R & D Systems; 1:200), and SOX2 Rabbit (Sigma; 1:500). Next, the tissue was washed four times thirty minutes, followed by overnight incubation at 4°C in secondary antibody in blocking buffer. Secondary antibodies were conjugated to Alexa flour anti-Mouse 488, anti-Rabbit 488, anti-Chicken 555, anti-Goat 647, or anti-Rabbit 647 (Life Tech; 1:1000). Nuclei were labeled using Hoescht Dye (1:2000), received as a gift from Bernd Fritzsch. Images were taken on either a Nikon C2 confocal microscope or a Leica Stellaris 5 confocal microscope and images were compiled in ImageJ and edited in CorelPhoto Paint (Version 19.0; 2017).

The cells were fixed and stained according to protocol (Kele et al., 2016 (link)). Briefly, fixation was performed in 4% PFA for 10 min, blocked in 10% bovine serum/0.2% Triton/PBS for 1 h before the primary antibody was applied diluted 1:200 in blocking buffer at 4°C overnight. Primary antibodies: Oct-4A-rabbit (Cell Signaling technology, #2840), Nanog-rabbit (Cell Signaling technology, #4903), SOX2-rabbit (Sigma-Aldrich, #AB5603), E-Cadherin-mouse (BD BioSciences, #610181), PLZF-rabbit (Thermofisher Scientific, #PA5-29213) and Nestin-mouse (Merck, MAB5326). All secondary antibodies were diluted in PBS in 1:500 using blocking buffer. Secondary antibodies: Goat anti-mouse-Cyanine3 (Thermofisher Scientific, #M30010), Donkey anti-rabbit-Alexa fluor 488 (Thermofisher Scientific, #A-21206). Nuclei were stained with DAPI (Life Technologies, #D1306), diluted 1:5,000 in PBS for 30 min. Image acquisition was performed on a Axioskop 2 (Zeiss) and software Axiovision version 4.8.

Whole mount immunohistochemistry was performed on previously fixed tissue^{42 (link)}. Ears were washed in phosphate buffered saline (PBS), then washed five times five minutes in PBS/0.05% Tween20 followed by blocking for one hour in 5% normal donkey serum, 1% bovine serum albumin, and 0.5% TritonX-100 in PBS. The tissue was incubated in primary antibodies, diluted in blocking buffer, at 4 °C for three nights. The following primary antibodies were used: MYO6 Rabbit (Sigma; 1:1000), MYOSIN7A Mouse (DSHB; 1:200), MYSOIN7A Rabbit (Proteus Biosciences, Inc.; 1:500), Neurofilament 200 (NF200) HC Chicken (Aves; 1:200), and SOX2 Rabbit (Sigma; 1:500). Next, the tissue was washed four times thirty minutes, followed by overnight incubation at 4 °C in secondary antibody in blocking buffer. Secondary antibodies were conjugated to Alexa flour anti-Mouse 488, anti-Rabbit 488, anti-Chicken 555, anti-Goat 647, or anti-Rabbit 647 (Life Tech; 1:1000). Nuclei were labeled using Hoescht Dye (1:2000), received as a gift from Bernd Fritzsch. Images were taken on either a Nikon C2 confocal microscope or a Leica Stellaris 5 confocal microscope and images were compiled in ImageJ and edited in CorelPhoto Paint (Version 19.0; 2017).