Inactivation and Clearing

Inactivation Protocol

Once you have a SWITCH tissue (4% PFA and 1% GA fixed tissue), you need to inactivate the tissue. Inactivation means terminating any fixation reactions still going on within the tissue. If you don't do inactivation, you might find a lot of background signals after labeling because antibodies become fixed to the tissue.

For inactivation, you will need:

Make a 4%(w/v) acetamide, 4%(w/v) glycine, 1X PBS solution and titrate it to pH 9 with NaOH.

Now you're ready to inactivate some tissues. But before you do that, you want to make sure the tissues are free of excess fixatives. You don't want to start fixing glycine and acetamide everywhere.

So, after fixation via either perfusion or SWITCH, the sample must be washed in PBST to remove unbound fixative molecules. For mouse brains, 2 washes of 6 hrs each at RT with gentle shaking was sufficient.

Then, finally, to inactivate remaining fixative molecules, you can incubate the sample in inactivation solution at 37 ˚C overnight (O/N for short). Then check the sample in the morning. If the solution turned yellow, the inactivation solution should be replaced with fresh solution and the sample incubated for several more hours. (If the sample needs to be cut, this should take place now before the sample is cleared.)

Clearing Protocol

We clear tissues with SDS. Briefly, SDS goes into the tissue and takes out the light-scattering lipid bilayers, making the tissue more transparent. This can be a time consuming step for large tissues.

One way to speed it up is to use a warm SDS solution. We speed it up more by using a hot SDS solution. Thanks to the stable SWITCH fixation (4% PFA, 1% GA), the tissues remain pristine even at high temperatures.

One problem though -- the tissue becomes brown at high temperatures. This is because of the Maillard's reaction, the same reaction that turns buttered bread into a golden-brown toast. We overcome it by using sodium sulfite, an antioxidant known to suppress this reaction.

For clearing, you'll need:

The clearing solution consists of 200 mM SDS, 20 mM sodium sulfite, 20 mM boric acid, and 10 mM sodium hydroxide. The pH should be around 9. Read this for more details.

Inactivated samples must next be incubated in clearing solution to wash away remaining inactivation solution and to distribute sodium sulfite through the sample. After 2 washes of 6 hrs each, the samples should be placed in a tube of fresh clearing solution, which should then be placed in a water bath heated to 70 ˚C. Other temperatures or methods of consistent heating may be used, but samples may deteriorate over time at higher temperatures. If a sample contains fluorophores that were genetically-encoded, introduced through viral injection, etc., then the sample may be cleared at 37 ˚C to preserve this fluorescence. The clearing process will take much longer at this low temperature, but temperatures higher than this will result in loss of fluorescence during clearing. Falcon tubes can become fragile over time in these conditions, so it is necessary to frequently check that the tubes have not begun to leak.