Dear colleagues,
I am trying to quantify astrocytes, neurons, and microglia in cortex and hippocampus. I would like to know how big should be the stack, for example, 20um??, the ideal objective and also how big should be each slide of the stack.
My sections are floating sections, 40 um big.
I appreciate your help.
Best,
Carolina
Hello Carolina! How are you planning to quantify this? What kind of samples (specie) do you have? If you have in mind that the small cell bodies are around 10um (microglia), you can choose have an overlapping interval (smaller than 10um) but be aware the cells can appear in more than one session. In this situation you can also check in 3D. In this way you will be sure you are not missing anything. In the other hand, if you don't want to risk to count the same cell twice, you can have a bigger interval, larger than 20 um (because neuron soma can get larger than this, depend of your sample). But your slice is already thin (40um). So, you don't have so much space to play around. If it is live samples, go for larger stacks to preserve the sample. If are fixed and you are not quantifying fluorescence intensity, go for smaller intervals (5um, for example) to not lose anything. You also can discard the first and last 10 um, imagining that those cells will be damage by the cutting... So, I suggest you to check (in the literature or in your actual samples the size of the structures (cell bodies? processes? some protein in the membrane, some organelle?) you are measuring and decide by that. Good luck!
Hi, dear people! thanks for your answer, now I looked and the literature and I found that a z-stack 20um long is ok, with 20 sections. I haven't quantified that yet.
At least I can take pictures of 20 um z-stack long.more than that it is impossible, I begin to see black above and below. As Paolo suggested I will use 40x magnification.
Thanks for sharing this information and helped me.
Best,
Carolina
Nice Carolina! But you mentioned before that your sample were 40um thick, is that right? Than if you use 20um z-stack you don't have room to acquire more than 2 z-stack (ideally just one at the middle)...
Hi Carolina,
How thick your stack is depends on HOW you quantify.
For instance, if you are looking at branches and the morphology of the cell, then you need to include all branches attached to the soma which you can see going up and down the z-plane using a confocal microscope because the branches are not all in the same place and you need to be sure which branch is attached to which cell. I would say that for this analysis you DO NOT need to have the same z-plane for all cells because some cells will have more/less complexity and therefore reach a difference distance from the soma.
On the other hand,iIf you are doing a simple fluorescence intensity count, then you just need to have the soma visible and there is no need to acquire a stack (no z-plane stack would be necessary) though if you do, because you are now quantifying fluorescence, the stack must be the same between cells/slides. Let me know if this is clear.
Cheers!
Thanks Tanya for your explanation, it is very clear!
Finally I did 15 um Z stack, one person in the lab suggested me to quantify positive area, but anyway I think I have to analyze morphology. I let in standby this experiments, if I have further question I will write for sure.
Thanks all of you for your help
With kind regards,
Carolina
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