The antibody has a proven and strong Biotin-Streptadvidin/HRP staining at a 1/2000-1/4000 dilution in rat amygdala, and spinal cord. The specificity of the antiserum was evaluated by soluble pre-adsorption with the peptides in question at a final concentration of 10-5M. CGRP immunolabeling was completely abolished by pre-adsorption with rat α -CGRP and partially eliminated by pre-adsorption with rat Ÿ -CGRP. Pre-adsorption with the following peptides resulted in no loss of immunostaining: rat amylin, rat adrenomedulin, calcitonin, neurotensin, somatostatin, substance P, leucine enkephalin, methionine enkephalin, VIP, CCK-8, vasopressin and neuropeptide Y.
Photo Description: IHC image of the dorsal horn of the rat spinal cord staining for CGRP (above) and low magnification image of a coronal spinal cord section staining for CGRP (below). The tissue was fixed with 4% formaldehyde in phosphate buffer, before being removed and prepared for frozen sectioning. Sections were incubated at RT in 10% goat serum in PBS, before standard IHC procedure. Primary antibody was incubated at 1:4000 for 48 hours, goat anti-rabbit secondary was subsequently added for 1 hour after washing with PBS. Light microscopy staining was achieved with standard biotin-streptavidin/HRP procedure and DAB chromogen.
Host:Rabbit
Quantity / Volume: 100 µL
State: Lyophilized Whole Serum
Reacts With: Bullfrog, Cat, Dog, Hamster, Human, Mouse, Mudpuppy (Necturus Maculosus), Ovine (Sheep), Pig, Platypus, Ram (Sheep), Rat
Availability: In StockAlternate Names: anti-CGRPRRID: AB_572217Immunogen: rat alpha-CGRPGene Symbol: CalcaNCBI Gene Aliases: CAL6,CGRP,Cal1,Calc,RATCAL6,calcitoniDatabase Links:Entrez Gene: 101095582 CatEntrez Gene: 403946DogEntrez Gene: 100772276 HamsterEntrez Gene: 796 HumanEntrez Gene: 12310 MouseEntrez Gene: 100294596 Ovine (Sheep)Entrez Gene: 100579174 PigEntrez Gene: 100579174 PlatypusEntrez Gene: 24241 Rat
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血清内含有抗体,封闭的时候,选用同物种的血清,比如,使用小鼠单抗的话,就选用小鼠血清来进行封闭。这样,如果细胞表面有可以非特异性结合小鼠抗体的部位都会事先被结合,就不会再结合荧光标记的特异性抗体了。
如果一个抗体可以用来“做western blot,免疫荧光,免疫共沉淀,ELISA”,我估计着是HRP或碱性磷酸酶或生物素之类标记的,然后需要再加荧光标记的二抗,这类抗体也是不能做流式用的。
我只用过流式的抗体来做免疫荧光(直接染色),效果还不错,但是反过来就没试过。
比如,你使用了小鼠IgG1, PE 标记的特异性抗体,那就选用小鼠IgG1, PE标记的非特异性抗体。
下面这个图,是一个比较典型的使用了同型对照的结果图向左转|向右转灰色的曲线是未染色的样本,黄色是同型对照,红色是特异性抗体染色。由于细胞对抗体的非特异性结合,一般同型对照都会比未染色样本的荧光信号要强。所以设置阴性阈值的时候,要根据同型对照,而不是未染色的细胞。