Hello everyone,
I am working with mammary fat tumors from mice. My goal is to characterize tumor-infiltrating immune cells between different tx groups. I did immunofluorescence staining with primary antibody e.g., CD4 and CD8, then incubation with secondary antibody e.g., Alexa 594, and DNA staining with DAPI.
For image analysis I am using imageJ software. I would like to count CD4 or CD8 positive cells in imageJ rather than manually counting but am struggling. I searched in the internet for any possible solutions but I did not find anything that could help me. Is there anyone who is experienced and can probably assist me with this please. I attached an image taken at 10X.
There are few ways to solve this and proceed with quantification. Although in ImageJ it is possible to count the number of cells automatically using plugin, Cell Counter (see attached files, how to do it). You might also encounter errors and miscalculations from the software etc.
However, the most reliable and scientific way to do such quantification is to do the following:
1. When we talk about the intensity or expression level of CD4, CD8, the magnification at 10X is ok and you can use Intensity per area to compare the control and test groups.
2. Here, when we talk about the number of cells, for some images 10X is OK. But, I would suggest to go a bit higher such as 20X or 40X. May be, try with 20X first and take 5-10 images from the same sample, with at least 3 biological replicates. In this case, you will end up with a relatively less no. of DAPI to divide for number of cells. Here, you can use another person as a blinded test to count the cells apart from you.
3. In any case, if the result is reproducible with manual or automated counting, with another person quantifying another dataset, you are good to go. I prefer such manual counting over ImageJ plugin, specifically for such tumor datasets.
Disclaimer for the attached pdfs - the files are accessible from a quick google search and the respective owners are in the pdf files. I don't own anything.
Good Luck.
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