Hi everyone,
so to quantify the reactivity of microglia, I would like to measure how much of the Iba1 signal is covered by CD68 by using IHC in mouse brain slices imaged with confocal microscopy at 40X magnification. Since the difference between samples is evident by eye already, I would like somehow to quantify that but I have more or less no clue on how to do it :( I was more thinking of setting as ROI the full area of the confocal images and not single cells. Is something like this possible at all actually? What would your suggestions be?
Thank you in advance for your help :)
Best Regards,
Marsela
Edit: here is an exemplary image. all my CD68 signal is located in the Iba1, but what I would like to measure is how much of the Iba1 is covered by CD68.
Hi Marsela Hakani,
If you could share an image you are working with we might be able to help.
Hi Marsela Hakani !
There are software tools available for this type of analysis. Fiji (ImageJ) is a free and commonly used tool that you might find useful. It allows you to set a region of interest (ROI) and calculate the degree of colocalization between two signals.
You can use the "Coloc 2" plugin within Fiji to quantify the correlation between the Iba1 and CD68 signals. The plugin provides several measures of colocalization, including Pearson's correlation, which may be useful for your analysis.
To use this tool, you would first set your ROI (which could be the entire image if you choose), then apply the "Coloc 2" plugin to that ROI. You can then compare the degree of colocalization across your different samples.
I hope this helps, and feel free to reach out if you have more questions!
Saman Behboodi Tanourlouee Thank you a lot for your answer, the coloc was something I thought of actually, but my only concern there would be whether this is the right method since at the end all of my Iba1 are CD68+ and vice verse, the only thing that changes are the expression levels. I will actually try it ;) Thank you.
Marsela Hakani Thank you for reference images. From the looks of your images, you could try thresholding method and then analyze the amount of area which is being covered. You could perform the following steps:
1. Convert the image in to 32-bit; Image>Type>32-bit.
2. Threshold the image; Image>Adjust>Threshold.
3. Convert the image to binary (B/W); Process>Binary>Make Binary. Be careful foreground (which will be quantified) is black and background is white, if this is not the case try inverting LUT; Image>Lookup Tables>Invert LUT.
4. Set the measurements as required (Area, SD, Mean are recommended) and check limit to threshold; Analyze>Set Measurement.
5. Analyze particle by Measure or Analyze particle function; Analyze>Measure; Analyze>Analyze particles.
This should leave you with good enough quantified data to compare treatments. If this worked, please respond.
If you are still unclear about the area quantification process, you could refer our recent publication.
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