I am working on immune reaction in Alzheimer's disease in mouse models at UCL. Currently I am doing co-immunostaining with antibodies against Iba1 (green) and CD68 (red).
My question is:
How to evaluate whether the microglia cell is activated or not? The signal varies quite highly, and can be either punctuate or globular, either in the cell body or in the processes or both.
As it is a phagocytocic marker I am aware that there is variation. However I am unsure of where to draw a line between unactivated to activated.
Thank you for advice!
It would be a different technique, but could you dissect mouse brain and do qRT-PCR with several microglial markers? TaqMan assays for all or most of them are available.
As about co-immunostaining, are you using control mice? Do you see any difference between control and experimental mice?
Hi!
This article shows a novel method to quantify activation by a cell size/soma size ratio, and also contains a lot of relevant references for traditional shape definitions for each of the 5 classical stages.
http://dx.doi.org/10.4103%2F2347-8659.139719
Activated vs. resting just via CD68 is not a black vs. white distinction. If you wanted pure counts you could simply count # of cells with CD68, or quantify the signal intensity or area of signal, although like you said as phagocytic marker none of those measures is very meaningful. If you just want simple analysis like this though I would recommend pairing it with quantitative analysis of microglia morphology using program like IMARIS (see links Elmore et al., 2014; Marsh et al., 2016).
Even better analysis might be to quantify what the microglia are phagocytosing and whether there is a difference. Several groups have utilized this protocol for examining amyloid phagocytosis (See links Guillot-Sestier et al., 2015; Marsh et al., 2016).
http://www.sciencedirect.com/science/article/pii/S0896627314001718
http://www.sciencedirect.com/science/article/pii/S089662731401201X
http://www.pnas.org/content/113/9/E1316.full?sid=dc554c59-0cf2-4446-9879-bf9bc10119b1
Hello,
I know it's been a long time that you have posted this question, but I am currently doing IHC on hippocampus tissues (alzheimer model). I want to use Iba1 and CD68 but I dont find a good CD68 antibody to do as a co-staining with iba1 because I am using anti-rabbit for iba1 (from Wako, the best one i think), so I need antother CD68 antibody (not rabbit). Can you please tell me how did you do your co-staining iba1 CD68?
Thank you very much!
yes it's mouse tissues. It's work well? I have to look for a good protocole for this co-staining!
Thank you Priya Prakash
anyways!
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