Sc 271546

For immunohistochemistry (IHC), patient tissues were fixed in 4% Paraformaldehyde, embedded in paraffin, and then cut into 5 μm thick sections for IHC staining. The slides were blocked with non-immune goat serum and incubated with anti-mouse GFRA1 antibody (1:200 Santacruz #sc-271546) for 24 h at 4°C. Then follow the instructions (ZSGB-BIO #SP-9002, Beijing, China).
For immunofluorescence, the transfected HCT116 cells and SW480 cells grown on the cover glass in 24 well plates were fixed with 4% paraformaldehyde (PFA) for 20 min and permeabilized with 0.2% Triton X-100 for 15 min at room temperature (22–25°C). Then, the cells were incubated with the desired primary antibodies in PBS for 24 h at 4°C, washed with PBS 3 times, and then incubated with fluorescent secondary antibodies away from light at room temperature (22–25°C). After that, the nuclei were stained with DAPI. The fluorescence intensity and absorbance were measured at 555 nm (Invitrogen #A21428, MA, USA) and 488 nm (Invitrogen #A11001, MA, USA), respectively.

Immunofluorescence staining of testes and SSCs was conducted as previously reported [27 (link)]. The primary antibodies used in this study are listed as follows: rabbit anti-eIF2γ (1 : 200; PA5-31177, Thermo Fisher Scientific, MA, USA), rabbit anti-DDX4 (1 : 200; ab13840, Abcam, Cambridge, UK), mouse anti-ZBTB16 (1 : 200; sc-28319, Santa Cruz Biotechnology, CA, USA), rabbit anti-STRA8 (1 : 200; ab49602, Abcam, Cambridge, UK), mouse anti-GFRa1 (1 : 200; sc-271546, Santa Cruz Biotechnology, CA, USA), rabbit anti-SOX9 (1 : 200; ab185230, Abcam, Cambridge, UK), and rabbit anti-StAR (1 : 100; bs-20388R, Bioss, Beijing, China). Secondary antibodies are as follows: Alexa Fluor 488-goat anti-rabbit IgG (1 : 400; ZF-0511, ZSGB-BIO, Beijing, China) and Alexa Fluor 568-goat anti-mouse (1 : 400; ZF-0513, ZSGB-BIO, Beijing, China).