In Situ Hybridization and Immunostaining of Xenopus Brain

Abstract

The dynamic expression pattern analysis provides the primary information of gene function. Differences of the RNA and/or protein location will provide valuable information for gene expression regulation. Generally, in situ hybridization (ISH) and immunohistochemistry (IHC) are two main techniques to visualize the locations of gene transcripts and protein products in situ, respectively. Here we describe the protocol for the whole brain dissection, the in situ hybridization and immunostaining of the developing Xenopus brain sections. Additionally, we point out the modification of in situ hybridization for microRNA expression detection.

Endothelial Brg1 fine-tunes Notch signaling during zebrafish heart regeneration

“…To generate RNA probes, we amplified notch1a , notch1b , notch2 , and notch3 cDNA from regenerating hearts at 7 dpa, blunt-ligated cDNA into a pEASy-Blunt vector, and generated digoxigenin-labeled RNA probes using T7 RNA polymerases (M0251; NEB). In situ hybridization was performed on cryosections of 4% paraformaldehyde-fixed hearts as previously described 68 .…”

Endothelial Brg1 fine-tunes Notch signaling during zebrafish heart regeneration

“…To generate RNA probes, we amplified notch1a , notch1b , notch2 , and notch3 cDNA from regenerating hearts at 7 dpa, blunt-ligated cDNA into a pEASy-Blunt vector, and generated digoxigenin-labeled RNA probes using T7 RNA polymerases. In situ hybridization was performed on cryosections of 4% paraformaldehyde-fixed hearts as previously (Liu, Wang, Li, He, & Liu, 2014).…”