Zebrafish in situ hybridization - formamide and tRNA (May/15/2008 )

Hi all,


I have a few questions regarding to zebrafish whole mount in situ hybridization, hope someone can help me. I'm confusing with too many different protocols available online.


1) Deionized formamide or formamide in hybridization buffer? Why should we use deionized formamide but not formamide?


2) I purchased yeast tRNA from Sigma in powder form. Should i extract RNA from it or use it directly in hybridization buffer?


3) How to store DIG-labeled RNA probes (600 bp) correctly so that it will last long? Is RNase needed in probe storing? Should i add the probe in hybridization buffer once it is synthesized to store it?


4) If the sense sample has a same staining pattern as the antisense sample, what does this mean? (I'm sure i didn't mix up both anti and sense probes and I got same result after repeating twice. I got my staining after overnight which is weird as normally i get my staining within 1-2 hours. Nothing change here. Could it be probe degradation?).


5) I need to perform ISH in various stages of embryos. Other than different proteinase K treatment time, any other parameter i should change to optimize for each stages?



Thanks in advance. Sorry for taking your time but I really have lots of questions for ISH.


Kuahmk

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http://search.vadlo.com/b/q?keys=zebrafish...mp;sn=158621799

I'm confusing with too many different protocols available online.
Thanks in advance. Sorry for taking your time but I really have lots of answers for ISH.

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formamide tends to break down, especially in the presence of moisture. deionization will remove the breakdown products (formic acid, ammonia). if you prepare your buffer fresh with deionized formamide then the formamide will not have enough time to break down too much.

the breakdown products can quench some fluorophores.

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