Novak Alicia E, Ribera Angeles B
Department of Physiology and Biophysics, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
Methods Cell Sci. 2003;25(1-2):79-83. doi: 10.1023/B:MICS.0000006894.43940.b1.
Two methods are presented here that allow clear visualization of antibody localization in zebrafish whole mount preparations, both for immunocytochemistry (ICC) alone and in combination with in situ hybridization (ISH). The first protocol describes ICC performed using a modified permeabilization technique and the chromogen AEC (3-Amino-9-ethylcarbazole). The second protocol describes the co-localization of transcriptional and translational products using a combined ISH/ICC protocol. A fluorescing chromogen (Fast Red, FR) is used to detect mRNA transcripts by ISH, and is combined with ICC that uses a secondary antibody conjugated to a different fluorescent molecule (Alexa 488). These procedures allow the identification of gene expression patterns in cell types identifiable with known antibodies.
本文介绍了两种方法，可清晰观察斑马鱼整装标本中的抗体定位，这两种方法既可单独用于免疫细胞化学（ICC），也可与原位杂交（ISH）结合使用。第一种方案描述了使用改良通透技术和显色剂AEC（3-氨基-9-乙基咔唑）进行的ICC。第二种方案描述了使用ISH/ICC联合方案对转录和翻译产物进行共定位。一种荧光显色剂（固红，FR）用于通过ISH检测mRNA转录本，并与使用与不同荧光分子（Alexa 488）偶联的二抗的ICC相结合。这些程序可用于识别已知抗体可识别的细胞类型中的基因表达模式。
