Handeling large numbers

SVSUNEURO

How long can the tissue (our sample is rat brain hemispheres) sit (post-perfusion) in the hydrogel solution prior to beginning the hydrogel tissue embedding process. Doing a handful of brains at once isn't an issue, but once we ramp up to full projects, 50+ animals at a time, there is going to be a workflow issue that we are going to have to manage. Is it best to let the sample sit in the hydrogel solution or should we transfer it to something else if it is going to sit for 4 weeks prior to embedding? Is it better to do a traditional perfusion (4% PAF) and then let the sample sit in 4% PAF until we are ready to begin hydrogel embedding? Other thoughts/options/suggestions greatly appreciated.

LauraLynch

I tried that and I might have ruined a bunch of brains in the process. We're not sure, but it's looking like extended time in hydrogel/paraformaldehyde is quenching our GFP signal.

A better option might be to embed all the samples and move them to clearing buffer. I've been doing that with my more recent samples.

If you keep the tissue at 37 degrees in clearing buffer, and change the buffer every day, the tissue starts to clear without electrophoresis. I've been doing that with my most recent samples for two weeks now.

SVSUNEURO

Hmm, are you hinting that, though the electrophoresis may speed the clearing process up, it might be easier (less risk to damaging the sample) to just let the sample clear without electrophoresis? It would take more time...but...everything is going to have to slow down once you get to the microscope anyway. Any risks associated with letting it sit in the clearing solution for too long?

To be honest, I would want my samples to all receive the same processing / timing / etc. So even if it takes a little more time to clear without electrophoresis, having all brains be in each wash/stage the same amount of time, mount for imaging (again, as long as the sample doesn't degrade once in focus clear). Methodologically makes the most sense to me.

Kristin_Engberg

Doing the perfusion with the hydrogel solution is definitely preferred over fixing the sample and then incubating in hydrogel solution later. If you are doing rat brains, I would suggest increasing the amount of hydrogel solution you use to perfuse - replace the traditional amount of 4% PFA solution with hydrogel solution (if you aren't already doing this).

I would also suggest embedding your samples after just 2 or 3 days sitting in hydrogel solution and then keeping them in clearing buffer. They should be stable in the clearing buffer for a few months at least, and it will probably take several weeks, but they will slowly clear over time from passive diffusion of the SDS into the tissue.

I wouldn't think that clearing your brains over a long time vs. using ETC should create significant differences such that you need to be anal about every sample receiving the exact same treatment. It is probably more important to make sure they sit in the hydrogel solution identical amounts of time and are embedded the same.

We would not recommend mounting your sample for imaging until the day or two before you are ready to image. We have found that FocusClear is not a good solution for storing the sample. After several days, we have seen precipitation forming in the tissue and it eventually turns opaque again. We aren't sure what this is - possibly crystallization? Either way, keep the samples in PBS (they should be stable for a long time) and then place in FocusClear.