Image Analysis (Fiji); immunofluorescence

Hi everybody,

I am performing immunofluorescence assays in my lab. I would like to study protein expression of different glial markers, and so its possible variation among my experimental groups. So, I am trying to find the correct method to analyze flourescence intensity of my samples (I downloaded the program fiji and cell profiler). I would be grateful if somebody could clear me what are the steps to follow for a correct analysis, what data I could get from my images?

Two example images are attached below (They represents AQP4 in red and GFAP in green, two common astrocytic markers. Cell nuclei are blue). The entire picture represents the region of interest.

Thank you for time spent!

Hi @Giorgia_Menegoni,

In case you are using CellProfiler, the workflow could be something like this,

  1. Nuclei is identified as Primary object using “IdentifyPrimaryObject” module.
  2. Using the Nuclei identify the corresponding cells with “IdentifySecondaryObject” module.
    Do this for both of your AQP4 & GFAP images.
    Some tips/suggestions,
  3. In GFAP channel you could Enhance the signal using “Enhance/Suppress features” module before segmenting the cells from GFAP image.
  4. Once you segment you can extract number of features using various measurement modules.
    For you to quick start with, example tutorials here. for ex. basic pipeline Human Cells pipeline.
    Hope this helps.

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Dear Giorgia,
usually to detect the fluo with fiji we use some workflow like

  1. Open the dataset
  2. split the channels
  3. Duplicate the channel X
  4. Select X-1
  5. Apply median filter
  6. Background Substraction
  7. Apply a threshold
  8. Create mask
  9. Refine Mask
  10. Watershed
  11. Set Scale
  12. Set Measurements (like mean or median)
  13. Analyze Particles
  14. Apply ROI to channel X

The best way to start
Image Analysis with ImageJ/FIJI Workshop