recently，I have completed some frozen slices of mice brain using IHC staining of Iba-1 to visualize the activation of microglia.But I found that the microglia is too much,could someone kindly providing me protocols for quantification of microglia activation?Or which sofeware I can use and the detailed protocol.I have photoed all the slices with 10X10 microscope.Thank you very much！
Could you explain what do you mean by "But I found that the microglia is too much"?
Too much signal? You can reduce the antibody concentration. The other problem might be from the thickness of your samples where signal from below you focus affects the results.
Microglia cells are very abundant in the normal brain. Iba1 detects all microglia cells (and macrophages too), either resting or activated. Resting microglia have a stellate-ramified morphology while activated microglia is amoeboid. That's a way to tell between the two but you need at least to image at 20x. Otherwise a good marker for activated cells is CD68. To quantify cells you need 20 or 40X magnification and a DAPI counterstaining; then you can quantify easily manually using ImageJ .
Hi Xilin,
I agree with Roberto. ImageJ is a good choice for your quantification. You can easily measure the signal intensity of your ibal staining or the size of microglia cell body (two criteria for microglial activation) by using this software. Just as what Roberto said, CD68 is a good alternative. Normally, in the brain, only activated microglia expresses CD68. Resting microglia is almost devoid of CD68 expression. If you have other problems about the quentifications, contact me. Good luck for your experiments.
Hi Xilin,
For microglial analysis, you would need to use atleast 20X or 40X magnification. As Roberto and Yingjun mentioned that you could use ImageJ for quantification. What kind of model system are you working on? If there is a considerable amount of microglial activation, then you could use CD68 or ED1 as a marker for activated cells and then count Iba1-CD68 double positive cells instead. You could also analyze the intensity of CD68 staining as an indicator of microglial activation. In addition, you could analyze the microglial morphology as either ramified (small round cell body with thin multiple processes), intermediate (elongated cell body with thicker cellular processes) or round/amoeboid (round or amoeboid cell body with no processes). Hope it helps.!
Thank you Deepi,you are also an expert for mocroglia.I am working on mice brain.so if I want to analyze the intensity of CD68 staining,I should use confocal microscope and keep the same gain value while aquiring pictures?And could you recommend the CD68 or ED1 antibody I should use?
You are welcome Xilin. Yes, acquiring images in a confocal microscope would be a better choice where you can use the same laser power, gain and offset for a given marker for better comparison. I have used ED1 antibody from Abd Serotech (Cat. No. MCA1957) which works perfectly well for staining mouse tissue. Good luck!
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