How to use ImmunoRatio plugin on ImageJ

Hello. Please I need help on how to use immunoratio plugin on imageJ or any other method of quantifying tyrosine hydroxylase expression of dopaminergic neurons. Thanks


First - you can try contacting the author of that directly via this link to their paper:

Second - perhaps you can share with us some original image files (directly upload here or via a link to a file sharing site) and describe what you wish to measure in your analysis? Try to stay away from biological-jargon (we are all coming from different scientific backgrounds and might not know your science) - so use words like ‘green signal’ or ‘blue blobs’ or whatever… just keep it simple. Then we can better help…

Thank you so much for your response. Here is one of the images I want to quantify, tyrosine hydroxylase on substantia nigra.



So… again - I don’t know what you want to ‘quantify’… do you want to segment the light purple blobs? Or the darker brown signal? Also - what do you mean by ‘quantify’? Measure what exactly… area, signal, etc. or just count?

Dopaminergic neurons. The darker brown signals. What I really need now is just the count but any other info will definitely be appreciated. Thank you so much



To start - ideally - you shouldn’t be using jpegs in quantitative image analysis… storing images in this way corrupts pixel values. So if you can - always save original image files in the proprietary format or as TIFFs… you can read more on why that is here:
Too - you don’t want to be burning in scale bars on images that you are analyzing… you can always overlay them later on for figures, etc.

For your type of image - I would either use the Color Threshold tool or the Trainable Weka Segmentation plugin. Using TWS - I got decent segmentation just using the default settings:

From there - you can segment those brown blobs to count. Here are some helpful links that will help you do just that:

The workshop is the best place to start - as it goes over how to use TWS and do step-by-step segmentation. :slight_smile:

Wow! Thank you so much Sir. I really appreciate this, but if I get stock at any point, trust me I am soooo coming back to disturb you. I can be an Oliver twist sometimes…lol.
Thank you so much.