Hi! I am planning to do some whole mount immuno for mouse embryonic tissue. I read about ScaleA2 and it seems like a good yet cheap option, particularly as it will preserve fluorescent proteins whereas I don't think alternatives like BABB will.
My first question: For the ScaleA2 protocol, I have read that I will have to incubate my samples in sucrose solution, followed by imbedding in OCT compound, freezing and thawing prior to clearing with ScaleA2. I was firstly wondering what the point of freezing is, if you're going to thaw your samples anyway? And if freezing is not necessary, then why use sucrose at all?
My second question: at what point should one put on their antibodies? I was planning to immunolabel prior to putting the samples in ScaleA2, but won't the sucrose solution I incubated my samples in impair the diffusion of the antibodies?
Any questions would be greatly appreciated!
Hi Daniyal,
If you're going to do whole-mount immunos, there's no need to freeze and OCT embed. I have cleared samples with Scale for whole-mount. It's very simple, after immunostaining, you fix them for a minimum of 30 min in 4% PFA, wash in PBS and then just transfer to ScaleA2 for clearing (here the time will depend on the size of your sample). If you want even further clearing, after they sink you can transfer them to ScaleB4 (8M urea). Hope this helps, let me you if you need more info.
Ana
Ana Rolo , may I know, how do You proceed with tissue sectioning of the samples for ScaleS protocol? I cannot find full protocol from A to Z, starting from perfusion and ending at imaging of sample.
I would need to clear brain sample, but I need to have it done on 105 µm thick sections. Should I cut them before IHC and then clear, or IHC, cutting and then clearance?
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