Anti oct4 antibody

Microarray analysis of purified mouse PGCs was performed as described44 (link). Expression levels of ovo-like genes and other germline markers were extracted from the microarray data. Microarray data were deposited in GEO under Accession No. GSE82020.
To count PGCs in mouse embryos, we used null-mutant mice lacking Ovol2 exon 3, which encodes the first and second of four zinc-finger domains35 (link). Oct4-positive PGCs in mouse embryos were counted as follows. Embryos of early somite stage (E8.0–8.5) were dissected and immunostained with anti-OCT4 antibody (Santa Cruz Biotechnology) as described45 (link). Z-stack confocal images were taken from each embryo using a Zeiss 510, and optical slices were analyzed using the ImageJ software. OCT4-positive PGCs were detected, and their X-Y positions in each section were recorded. PGCs with the same (or almost the same) X-Y positions were considered to be identical, and total PGCs were counted by manual inspection of all optical sections.
All mouse experiments conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Committee of Laboratory Animal Experimentation of RIKEN BioResource Center.

Cells were lysed and sonicated in RIPA buffer, following SDS-PAGE of lysates. Proteins were transferred to Hybond nitrocellulose membranes (Amersham, GE Healthcare) and immunoblotted with anti-OCT4 antibody (1∶1,000; sc-5279; Santa Cruz Biotechnology), anti-LIN28B antibody (1∶1,000; #4196, Cell signalling) and anti-GAPDH (1∶10,000; #4300, Ambion). After washing with PBST, secondary HRP-linked antibody ECL Mouse IgG, (1∶5,000; Amersham) were used and detected with enhanced ECL reagent (Amersham). Figure 4D, Chemiluminescent Detection Films (Roche) and CURIX 60 film developing machine (Agfa) was used to visualize ECL-activity. Figure 3B, ECL-activity was detected with a FUSION-FX7 Advance Chemiluminescent system (Peqlab).