I've already done some tests with both BABB and iDISCO clearing, while with the first method at the end I obtain a tissue that has become harder and more resistant than it was at the beginning, with the second method the sample becomes yes a bit harder but also more fragile, the tissue breaks easily.

Since the parts of the brain that I have to analyze are not that big I'm afraid that, trying to secure it to the sample holder of the light sheet microscope that I'm using with the screw (because the holder works simply screwing a screw until the sample gets stuck), they will break. Agarose embedding has also been suggested by the technician working with the microscope.

So I'd like to do some test making small cubes of agarose of different percentage and see if they become properly transparent after dehydration and immersion in DBE and if they also become fragile.

I was thinking that I could dehydrate, then rehydrate and then immunolabel the samples and only at the end, before the last dehydration step, I could embed them in agarose. In this way, the Ab penetration is not affected.

I just have to be sure that the clearing in this way is not affected as well.

Dear Francesco,

Since this thread is relatively old, I would like to ask you if you finally gave a try to the agarose-embbeding prior the antibody incubation and clearing. I am thinking on trying it out and am curious about your experience.

For the agarose blocks, I have done it for having some "cushions" for the sample holder of the microscope, which sometimes become handy (as your technician suggested). I follow the same steps as for tissue clearing, except for the DCM incubation and extending every step of the methanol series (20, 40 , 60, 80, 100, 100) a bit longer (maybe 1.5-2h and the last 100% methanol for 1-2 days, changing it 1-3 times), before the immersion in DBE. In the past, I did use the same timepoints, but the blocks weren´t completely dehydrated and when immersed in DBE, it was causing that the solution was turning somehow milky (and of course, these guys had to be discarded).

Another way of fixing your sample to the holder is to use a very thin layer of Cyanoacrilate glue (Loctite or Krazy glue). Be very careful since it can destroy your sample if reaches areas of interest. Put the glue on the holder, make a very thin layer (I use a pipette tip), put your sample on top of the glue (the area of the sample in contacct wit the glue should be of no interest), cover your sample while drying for some minutes and fix it to the microscope holder. I found sometimes that adjusting the sample with the screw (we have Lavision Ultramicroscope) it was either causing some cracking in the tissue and leaving it too loose that I was ending up with my sample floating around in the imaging chamber.

Thanks in advance and all the best with your experiments.

Cheers,

Carlos