Mouse anti ki 67

Ki67 is expressed in actively dividing cells (all stages of the cell cycle except G₀) and therefore is expressed at higher levels than BrdU 24 h after injection of BrdU (Kee et al., 2002 (link)). Randomly selected brain sections from the same animal were also immunohistochemically stained with anti-Ki67 or anti-Sox2 (n = 8 per sex). Brain sections were prewashed with 0.1 m PBS and left to sit overnight at 4°C. The next day, sections were incubated in 10 mm sodium citrate buffer for 45 min at 90°C to retrieve antigens of Ki67 and blocked with 3% NDS and 0.3% Triton X-100 in 0.1 m PBS, followed by incubation in primary antibody solution made with 1:1000 mouse anti-Sox2 (Santa Cruz Biotechnology) or 1:250 mouse anti-Ki67 (Leica Biosystems), 1% NDS, and 0.3% Triton X-100 in 0.1 m PBS for 24 h at 4°C. Then the sections were incubated in secondary antibody solution, consisting of 1:500 donkey anti-mouse Alexa Fluor 488 for Sox2 (Invitrogen) and 1:500 donkey anti-mouse Alexa Fluor 594 for Ki67 (Invitrogen), 1% NDS, and 0.3% Triton X-100 in 0.1 m PBS, for 18 h at 4°C. After three rinses with PBS, sections were incubated in 1:5000 DAPI in PBS for 3 min and mounted onto slides and cover-slipped with PVA DABCO.

Embryos were harvested at E14.5, fixed overnight in 4% paraformaldehyde at 4°C, washed in PBS, dehydrated through a graded ethanol series, and stored in 100% ethanol at −20°C. Embryos were rehydrated in PBS, cryoprotected in 30% sucrose overnight at 4°C, embedded in Tissue-Tek OCT Compound (Sakura Finetek USA, Inc., Torrance, CA), quick-frozen on dry ice, and cryosectioned at 16 µm. Primary antibodies used for immunohistochemistry and their dilutions are as follows: mouse anti-neurofilament (1:250, 2H3), mouse anti-islet1/2 (1:100, 39.4D5), mouse anti AP-2alpha (1:100, 5E4) were obtained from Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA); rabbit anti-Pax2 (1:250, 71-6000, Invitrogen); mouse anti-Ki67 (1:1000, ACK02, Leica Biosystems). Detection of primary antibodies was achieved using secondary antibodies conjugated to Cy3 (1:100, 115-106-006, Jackson ImmunoResearch Laboratories) or Alexa 488 (1:100, A32723, Molecular Probes). Section in situ hybridization was performed with digoxygenin-UTP-labeled riboprobes essentially as described (Nissim et al., 2007 (link)). At least three to five embryos in the experimental and control groups were evaluated for each antibody or in situ probe.