Dear Shan Jin,

In general, the area of IHC and FISH image analysis is gradually moving to the automated quantification. However, acceptance of your results will depend on the rules of the particular journal you are submitting your manuscript to.

Below is a link to the one where the automated quantification was accepted. Please note the abundance of controls. Also, please remember that the main advantage of histology is detecting the location of the signal. So, when assessing the data, make sure you present the data in regard to the signal location within the cell of tissue. Good luck!

https://www.nature.com/articles/s41598-018-26345-0

check this link:

https://www.nature.com/articles/s41598-018-26345-0

Dear Shan Jin, if you substract background your approach is okay, although you can not speak of a real quantification.

Many thanks for all replies. In my second and third trials I was not able to obtain a nice correlation between ISH mean intensity and RT-qPCR results. I agree with Torsten that measuring intensity values from fluorescence staining is not a good method to achieve accurate quantitation.

I believe that reviewers prefer luminescence instead of fluorescence.

But as Irina said, fluorescence quantification it's slowly getting reliability among reviewers.

From my experience i have already obtained very confident results quantifying fluorescence in vivo from zebrafish larvae.

Good Luck

Filipe Ricardo Pires Carvalho Thank you! Is your probe directly conjugated with fluorophores or with other molecules for further signal amplification? I am using TSA amplification which I think somehow might affect linearity of the fluorescence intensity.

My experience comes from fluorescence quantification performed for mcherry signal in vivo. But if you have problems with linearity of your quantification the only solution is to increase the sample.

best