I have recently performed Thioflavin-S staining procedure to visualise amyloid plaques in the tissue sections of Alzheimer Disease mouse brain. Hence, I am looking for proper methods to quantitate the amyloid plaques in my images which I can use for further analysis. Can anyone help me in a good detail as to how can I quantitate the amyloid plaques and which software/tools to use?
Hi Apurva, this is a complex question..so, if you need to exactly quantitate Ab plaques in the brain, is quite time consuming, because you need to:
1-have a series of sections for the whole brain or at least that encompass the whole area (i.e cortex) of interest..so what kind of section? coronal? sagittal? etc...
2-choose a interval in which examine the section, for example, every sections or every three, or every five...depends on thickness of the section and how many of it you have
3-use a software by which you can measure plaque area and plaque number . I will suggest to use ImageJ from NIH with the plugin from Wright Cell Imaging Facility, it's free and easy to use, here is the link http://www.uhnresearch.ca/facilities/wcif/ Also in their website you can found additional suggestions on histological analysis
Hope this helps! enjoy!
Apurva,
I too vote for ImageJ with appropriate plugins is a great tool for quantification of your microscopic images. I would suggest you to look at figures 1, 6 and 7 of Mol. Pharmaceutics, 2012, 9 (7), pp 1887–1897 where ImageJ has been used for quantification of microscopic images, but for a different purpose.
This protocol from David Morgan's lab is a good resource (Nature Protocols 1, - 1591 - 1595 (2006).
Amyloid plaque analysis can be much simpler. If you want to report % area of cortex or hippocampus positive to cong-red stain then you can use Adobe Photoshop CS3 for both image processing and quantification together in the same action and automate the entire workflow. However, it is not freely available. Another great option is MBF Image J. One can also easily record a macro for automation purpose.
@Lakshman: Do you have a copy of this paper? I would be grateful if you can mail me the paper on: [email protected]
I use image J all the time for plaque quantification. You convert your images to 8 bit grayscales, open them up in image J and then outline the area of the brain you're interested in using their ROI tools. Then set your measurements, I use area, and then threshold your images to convert them to binaries. then usng the analyse particles function, you can get a number for the total area occupied by the immunostain in your area of interest in that section. normalise it to the total area measured, and you will end up with an area fraction for every section that you analyse. Sampling sections is really up to you and depends on what specific question you're asking.
The also convincing to add in addition a picture of an overall area at relatively low magnification.
The imageJ particle analyze works like a charm, I also agree.
I also found these papers super nice:
Sun, A., X. V. Nguyen, and G. Bing. 2002. “Comparative Analysis of an Improved Thioflavin-S Stain, Gallyas Silver Stain, and Immunohistochemistry for Neurofibrillary Tangle Demonstration on the Same Sections.” Journal of Histochemistry & Cytochemistry 50(4): 463–472. http://jhc.sagepub.com/content/50/4/463.full (July 13, 2012).
NeuroscienceVolume 105, Issue 1, 16 July 2001, Pages 99–107The morphological phenotype of β-amyloid plaques and associated neuritic changes in Alzheimer’s diseaseT.C Dickson, J.C Vickers,
Hi! I am also trying to use Image J to analyze amyloid plaques in monkey brain. The problem I encounter is that there is a lot of intracellular staining. How do you eliminate the intracellular staining from the analysis to count only plaques? Thank you!
Maria Luisa Appleman : You can use manual threshold to select only plaques. Follow this paper for detailed Thioflavin-S staining analysis procedure: "NLRP3 is activated in Alzheimer’s disease and contributes to pathology in APP/PS1 mice".
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