Protocol PDF 

0. Protocols at a glance

PDF file Contents Lat updated
<Basic Protocols>
floatingISHe.pdf Floating ISH with a single probe (color detction) 2007.8.30
DISHprotocol.pdf Fluorescent double ISH 2010.1.18
PCR_cloning.pdf Gene fragment cloning by RT-PCR 2007.8.30
ISHlogsheet.pdf Logsheet for single ISH 2007.8.30
DISHlogsheet.pdf Logsheet for double ISH 2007.8.30
TSAbiotin.pdf Method to make "In-house" TSA-biotin system 2010.10.19
BrdU_ISH.pdf Double staining for BrdU and ISH 2010.10.19
Methods.pdf A method paper in "Methods", Elsevier 2010.10.19


1. What each protocol explains

1) Basic protocol


A protocol for floating ISH.  Colorimetirc detection of a single probe hybridization using NBT/BCIP substrate.  Used for formalin-fixed frozen tissue.  


A protocol for fluorescent double ISH.  In this protocol, the fluorescent detction is enabled by (1) sensitive Alkalin phosphatase substrate, HNPP/FR and (2) amplification of the hybridization signal using TSA (tyramide signal amplification).  They can be easily implemented for most ISH methods.  You can find the principles and tips to use fluorescent detction.  


A protocol to construct a probe plasmid from RNA.  Covers Reverse transcriptase (RT) PCR reaction, subcloning of the PCR fragment into plasmid vecotrs, how to confirm the cloned fragments, template synthesis, in vitro transcription.  

2) Addition


Logsheet for single ISH


Logsheet for double ISH


Protocol for a homemade TSA-biotin;


A protocol to combine birhhtdating by BrdU and ISH.


The preprint version of the paper published in "Methods", Elsevier. In addition to normal dual-probe ISH, various fluorescent ISH methods are discussed, including tracer-ISH and BrdU-ISH double labeling .



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