Induction of c-Fos expression in mouse vomeronasal neurons by sex-specific non-volatile pheromone(s)
Induction of c-Fos expression in mouse vomeronasal neurons by sex-specific non-volatile pheromone(s)
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DOI：
10.1093/chemse/bjh156
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发表时间：
2005-01-01
期刊：
影响因子：
3.5
通讯作者：
Touhara, K
中科院分区：
文献类型：
--
作者：
Kimoto, H;Touhara, K
材料和方法 BALB/c 小鼠（SLC，静冈县，日本）饲养在 12 小时光/暗周期（上午 8:00 亮灯）下。成年雄性小鼠被单独放置在干净的垫料中。 2天后，收集污染的垫料并用于分析。通过握住尾巴并等待自然排出来从 BALB/c 成年雄性收集尿液。收集的尿液立即冷冻并保存在–80°C 下直至使用。将小鼠暴露于干净的垫料或弄脏的雄性垫料，并在连续暴露90分钟后处死。刺激在上午 8:00 至 11:00 之间进行。暴露后，用冰冷的 4% 多聚甲醛 (PFA) 的 PBS 溶液对小鼠进行心内灌注。取出口鼻，并在 4% PFA 的 PBS 溶液中于 4℃ 后固定 4 小时，然后在 0.5 M EDTA 中于 4℃ 下孵育 48 小时。将样品置于含 30% 蔗糖的 PBS 溶液中于 4℃ 下固定 20 小时，并包埋在 OCT（樱花，东京，日本）中。每三个冷冻切片（每个 15 µm）被收集并安装在 MAS 涂层的载玻片（Matunami Glass Ind. Ltd，日本）上。将载玻片在含有0.1% Triton X-100 (TBST)的TBS中用1% H 2 O 2 处理30分钟，然后与在TBST中含有3%牛血清白蛋白的封闭溶液一起孵育。将 VNO 切片与 1:5000 稀释的抗 c-Fos 多克隆抗体（Ab-5；Oncogene，圣地亚哥，加利福尼亚州）在封闭溶液中于 4℃ 孵育 60-70 小时，然后与生物素化山羊抗兔二抗（Vector Laboratories，伯林盖姆，加利福尼亚州）在室温下孵育 1 小时。使用ABC试剂盒（Vector Laboratories）和DAB（Sigma）进行染色。然后将染色切片与 1:500 稀释的抗 Gαo 抗体 (Santa Cruz Biotechnology, Santa Cruz, CA) 在 TBST 中于 4°C 孵育 24 小时。在共聚焦显微镜下，使用 Alexa488 缀合的山羊抗兔二抗 (Molecular Probes, Eugene, OR) 确定 c-Fos 阳性神经元的位置。
Materials and methodsBALB/c mice (SLC, Shizuoka, Japan) were housed under a 12 h light/dark cycle (light on at 8: 00 am). Adult male mice were placed individually in clean bedding. After 2 days, the soiled bedding was collected and utilized for assay. Urine was collected from BALB/c adult males by holding the tail and waiting for natural discharge. Collected urine was immediately frozen and stored at–80 C until use. Mice were exposed to clean bedding or soiled male bedding and were killed after continuous exposure for 90 min. The stimulation was carried out between 8: 00 am and 11: 00 am After exposure, mice were perfused intracardially with ice-cold 4% paraformaldehyde (PFA) in PBS. Snouts were removed and post-fixed in 4% PFA in PBS for 4 h at 4 C and then incubated in 0.5 M EDTA for 48 h at 4 C. The sample was placed in a 30% sucrose solution in PBS for 20 h at 4 C and embedded in OCT (Sakura, Tokyo, Japan). Every third cryosection (15 µm each) was collected and mounted on an MAS-coated glass slide (Matunami Glass Ind. Ltd, Japan). Slides were treated with 1% H 2 O 2 for 30 min in TBS containing 0.1% Triton X-100 (TBST), followed by incubation with a blocking solution including 3% bovine serum albumin in TBST. The VNO sections were incubated for 60–70 h at 4 C with a 1: 5000 dilution of anti-c-Fos polyclonal antibody (Ab-5; Oncogene, San Diego, CA) in the blocking solution and then incubated with the biotinylated goat anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA) for 1 h at room temperature. Staining was performed using ABC kit (Vector Laboratories) and DAB (Sigma). The stained sections were then incubated with a 1: 500 dilution of anti-Gα o antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in TBST for 24 h at 4 C. Under a confocal microscope, the locations of c-Fos positive neurons were determined using Alexa488-conjugated goat anti-rabbit secondary antibody (Molecular Probes, Eugene, OR).