for decreasing background during mRNA whole mount in situ In situ hybridization acetylation with acetic anhydride (0.25%) in triethanolamine is required. Is there any alternative to Acetic Anhydride which can be using along with triethanolamine as Acetic Anhydride is a banned chemical which is not available easily in our country.
Interesting question! Since ISH protocols have been around for a long time, they have surely also a complicated history. Often the significance of individual steps of the protocol are hard to understand. Although the acetylation step to reduce background seems on paper very important, it might be worth trying to do it without this step.
For example, the abcam protocol omits this step (http://www.abcam.com/protocols/ish-in-situ-hybridization-protocol). Or see also: https://lifescience.roche.com/wcsstore/RASCatalogAssetStore/Articles/BIOCHEMICA_98_1_p10-16.pdf or
https://vectorlabs.com/data/protocols/InSitu.Hybrid.Meth.pdf.
As I said, many "traditional" protocols have accumulated weird steps over time or included steps that may not be required anymore because today's reagents are much cleaner. We used to clean formamide to remove contaminants. I am sure this is not necessary anymore. If you look at older plasmid DNA miniprep protocols, they vary a lot and include steps that may not be needed.
Even some protocols form the old Roche ISH handbook go without an acetylation step (http://www.diagnostics1.com/MANUAL/In_situ.pdf).
Perhaps the RNAseA step has always taken care of unspecifically bound probe when using RNA probes? I only could find little on the exact purpose of the acetylation step:
"Another commonly observed pre treatment when using RNA probes is acetylation with acetic anhydride (0.25%) in triethanolamine. This treatment is thought to be important for decreasing background but it also appears to inactivate RNases and may help in producing a strong signal. Luckily RNases are not an issue when oligonucleotide probes are used. Fixation of tissue effectively "secures the target RNA" i.e. the mRNA within the tissue from RNAse digestion and oligonucleotide probes are naturally resistant to RNases. This is why solutions do not need to be RNase free when using oligonucleotide probes with in situ hybridization." (from: http://www.genedetect.com/insitu.htm).
Hope this helps.
Interesting question! Since ISH protocols have been around for a long time, they have surely also a complicated history. Often the significance of individual steps of the protocol are hard to understand. Although the acetylation step to reduce background seems on paper very important, it might be worth trying to do it without this step.
For example, the abcam protocol omits this step (http://www.abcam.com/protocols/ish-in-situ-hybridization-protocol). Or see also: https://lifescience.roche.com/wcsstore/RASCatalogAssetStore/Articles/BIOCHEMICA_98_1_p10-16.pdf or
https://vectorlabs.com/data/protocols/InSitu.Hybrid.Meth.pdf.
As I said, many "traditional" protocols have accumulated weird steps over time or included steps that may not be required anymore because today's reagents are much cleaner. We used to clean formamide to remove contaminants. I am sure this is not necessary anymore. If you look at older plasmid DNA miniprep protocols, they vary a lot and include steps that may not be needed.
Even some protocols form the old Roche ISH handbook go without an acetylation step (http://www.diagnostics1.com/MANUAL/In_situ.pdf).
Perhaps the RNAseA step has always taken care of unspecifically bound probe when using RNA probes? I only could find little on the exact purpose of the acetylation step:
"Another commonly observed pre treatment when using RNA probes is acetylation with acetic anhydride (0.25%) in triethanolamine. This treatment is thought to be important for decreasing background but it also appears to inactivate RNases and may help in producing a strong signal. Luckily RNases are not an issue when oligonucleotide probes are used. Fixation of tissue effectively "secures the target RNA" i.e. the mRNA within the tissue from RNAse digestion and oligonucleotide probes are naturally resistant to RNases. This is why solutions do not need to be RNase free when using oligonucleotide probes with in situ hybridization." (from: http://www.genedetect.com/insitu.htm).
Hope this helps.
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