Mouse anti plzf antibody

For α-GalCer stimulation, the sorted iNKT cells were seeded in a 96-well plate in DMEM (with 10% FBS), left unstimulated, or stimulated with α-GalCer (125 ng/ml) for 72 h. α-GalCer -stimulated iNTK cells were dropped on the poly-L-lysine slides, incubated for 30 min, fixed with 4% paraformaldehyde, and then permeabilized with 0.05% PB buffer. The unstimulated iNKT cells were incubated with a mouse anti-PLZF antibody (4 μg/ml, Santa Cruz Biotechnology) and AF488-Actin (Invitrogen) for 1 h, further stained with a Goat anti-mouse AF546 secondary antibody (1:400) for 30 min, and finally covered with 1.5 μg/ml DAPI (Beyotime). α-GalCer stimulated iNTK cells were incubated with rabbit-anti-FoxO-1 (Cell signaling technology) and AF488-Actin for 1 h, and further stained with a AF546-Goat anti-rabbit secondary antibody (1:400) for 30 min, and finally covered with 1.5 μg/ml DAPI (Beyotime). Images were collected and analyzed using a confocal microscope (Nikon A1R).

The testicular tissue fragments of the experimental groups, in addition to the tracing of DiI, were subjected to immunohistochemistry after tissue processing. To confirm the nature of SSCs, spermatocyte and spermatozoa the PLZF protein [17 (link)], SCP3 protein [20 (link)] and the ACRBP protein [21 (link)] were detected, respectively. The procedure of immunocytochemistry was performed according to previous study [22 ]. Briefly, tissues fixed with 4% paraformaldehyde (Sigma, USA) in PBS were Cryo-embedded in OCT compound (optimal cutting temperature) (Sakura, Japan) and cut into 5 µm-thick sections. Incubation with primary antibodies was applied for overnight at 37°. Then the second antibody was applied for 2 h at room temperature in the dark. Nuclei were counterstained with DAPI. Specimens were observed with a confocal laser microscope (TE 2000, Nikon, Japan). The following antibodies were used as primary antibodies: mouse anti PLZF antibody (1:100 Santa Cruz Inc, USA), Rabbit anti SCP3 antibody (1:400 Abcam, UK), Rabbit anti ACRBP antibody (1:300 Abcam, UK). The secondary antibodies used were goat anti mouse IgG and goat ant rabbit igG, conjugated with Alexa 488 (1:200, Bio legend UK).