Dab substrate reagent kit

Immunohistochemical staining using appropriate antibodies (Supplementary Table S2) was performed as follows. Tissue sections were mounted on glass slides, dewaxed, rehydrated and submitted to antigen retrieval by boiling for 20 min in a citrate buffer (pH 6). Endogenous peroxidase activity was blocked by a 10 min incubation with 3% hydrogen peroxide. Slides were then washed in PBS and blocked for 30 min in 2% horse serum. Slides were incubated overnight at 4 °C with primary antibody diluted in PBS, 20% blocking buffer. Bound primary antibody was revealed by 30 min incubation with peroxidase-conjugated secondary antibody (ImmPRESS reagent kit, Vector Laboratories) and finally with 3,3′-diaminobenzidine (DAB substrate reagent kit, Vector Laboratories). For immunofluorescence, 4% formaldehyde-fixed embryoid bodies (EB) or cell mixture aggregates (CMA) were submitted to antigen retrieval with citrate buffer (pH 6). Sections were then blocked either in 2% horse serum, and the slides were mounted with the Vectashield 4,6-diamidino-2-phenylindole (DAPI) medium (Vector Laboratories, Newark, CA, USA). Imaging was performed using a Leica DM5500 B epifluorescence microscope (Leica Microsystems, Wetzlar, Germany) equipped with a CoolSNAP HQ² camera (Photometrics) and Leica MMAF software (Metamorph). Images were processed with Image J software.

The gonads were fixed in 4% formaldehyde. The fixed gonads were dehydrated, embedded in paraffin, and cut into 5 μm thick sections. Sections were mounted on slides, dewaxed and rehydrated. For histological analysis, sections were stained with hematoxylin and eosin.
For immunohistochemical staining, sections were boiled for 20 min in citrate buffer after dewaxing and rehydration. Endogenous peroxidase activity was blocked by incubating sections for 15 min with 3% hydrogen peroxide. Sections were then blocked for 30 min with 2% horse serum and incubated overnight at 4 °C with primary antibody (see Table 2 for antibodies). After three washes in PBS, the slides were incubated with peroxidase conjugated secondary antibodies (ImmPRESSTM reagent kit, Vector Laboratories, Eurobio, Les Ulis, France) for 30 min at room temperature. Antibodies were revealed with DAB (DAB substrate reagent kit, Vector Laboratories) or VIP Vector (VIP substrate reagent kit, Vector Laboratories).