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: https://breast-cancer-research.biomedcentral.com/articles/10.1186/bcr2615
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: https://imagej.net/Principles#Why_.28lossy.29_JPEGs_should_not_be_used_in_imaging
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.
From there - you can segment those brown blobs to count. Here are some helpful links that will help you do just that:
- Segmentation page
- “Segmentation in Fiji” workshop and corresponding slides (more recently updated slides are here)
- Trainable Weka Segmentation (TWS) plugin and it is scriptable via macro code - a great tool for segmentation that comes directly with Fiji. NOTE: Fiji is Just ImageJ - it is simply a distribution of ImageJ that comes with a bunch of plugins bundled - ready for you to use out-of-the-box. If you are just getting started, we recommend downloading/using Fiji.
The workshop is the best place to start - as it goes over how to use TWS and do step-by-step segmentation.
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.