PACT and Immunos

bpham

I have been clearing tissue using the PACT protocol on 1mm sections of mouse spinal cord. I use 4% acrylamide and 8% SDS.

I incubate in primary for 3 days at room temperature at a concentration of 1:200
Wash in PBS for a day
Incubate in secondary for 3 days at room temperature at 1:200
Wash in PBS for a day
Put into RIMS for 2 days.

The tissue is not as transparent as tissue I put into RIMS after the SDS wash and 1 day PBS wash without running immunos.

Has this happened to anyone else?

DUJ

Yes, this happened to me as well. However, it also seems to happen if I just leave the sample in PBST for some days, so I think it is not the immuno itself causing the problem. I tried to find the cause for this (temperature, pH..) but did not succed so far. Strangely, it does not happen every time even though the incubation conditions are kept the same. I would be really interested to hear if anyone found a way to get rid of this issue.

dgroberts

@DUJ It is definitely the PBST that is the problem. The only way I have been able to avoid it is by washing in PBST several times over the course of only a few hours right after clearing, and then placing into an RI matching solution as soon as possible. The residual SDS that may or may not remain hasn't proved to be an issue (at least in RIMS), but has done wonders for my imaging depth (and overall tissue transparency) of mice brains.

But of course this is an issue if you're doing immunostaining and need to wash in PBST for several days. I haven't quite figured this one out yet, but I have a couple ideas in mind... First, you might want to try adding some RI matching solution to your samples while incubating. You might also want to try a different detergent (perhaps Tween-20 instead of Triton X-100) and incubating at a higher temperature. I know that @bpham is even trying a gentle wash with SDS (maybe try a 1 or 2% solution to avoid the elution of antibodies). Additionally, I was thinking of incrementally decreasing my [SDS] towards the end of clearing and placing my samples straight into RIMS with a lower initial [Histodenz] (for lower viscosity and better penetration). I would then simply increase its concentration until I have the correct refractive index. For whatever reason, Histodenz seems to prevent SDS's precipitation well at room temp.

DUJ

@dgroberts Thank's for your input, today I immersed several samples in different buffers with different detergents (Triton, Tween, NP-40) to see what gives the best results. Will get back with the results.

dgroberts

@DUJ Great! Please let me know how that goes.

DUJ

@dgroberts The problem seems to be the PBS, samples washed in Borate buffer (0.2M Boric Acid, pH to 8.5) are much more transparent after 5 days. No difference between detergents.

dgroberts

@DUJ Great news, thanks for the info! Looks like I'm going to have to give the borate buffer a try!

Adam

Not sure if this is helpful, but I haven't found PBS to be any problem for transparency, as long as I'm very careful to wash out any residual SDS after clearing.

bpham

Sorry I haven't checked up on this lately. I believe there is a bigger transparency issue for doing spinal cords rather than he brain, especially for immunos. I did take some old 1 mm spinal segments that I did immunos on. I first post-fixed this tissue for two hours and put it back into 8% SDS at 40 C for 24 hours. I then washed in PBST for 24 hours and put back into RI solution. The tissue became crystal clear again. When I imaged I did notice less antibody antigen attachment but it did wonders for my background issue.

bpham

I'm currently waiting on a new set of tissue to further test what happens when I put my tissues back into differing SDS concentrations with or without post fixation after immunos to see how much clearer my tissues become and to minimize antibody detachment. @DUJ, I will have to try that, thanks!!