Gel polymerization does not work

jimichu

Dear forum members.
I have been having problems with the HYDROGEL polymerization. Do you think it is necessary to fill up the vacuum chamber with nitrogen after degassing?. So far I was not doing it and gels were polymerizing OK but with inconsistent results.
I would like to know if there is a clear and consistent way of getting HYDROGELS. Maybe it has to do with the reagents, I am using homemade 16% PFA and the rest of the reagents are commercial ones.

Thanks in advance, any suggestion would be greatly appreciated.

liz_kritzer

I also had some problems getting hydrogel polymerization early on. Based on my experience, I think it is necessary to fill the chamber with nitrogen after degassing. It's very important to have little or no oxygen in your sample. If you fill the chamber with air instead of nitrogen, you will be introducing oxygen again. Also, I had mixed results with homemade PFA. I have had more consistent results with the commercial 16% PFA listed in the manuscript.

shlomilazar

Dear Liz,
Please try the following setup: I fill the 50ml tube (with the hydrogel and brain) with N2 using a syring niddle that is connected to N2 container. I also puctured the lid nearby to let N2 flow to get out. I kept it running for 30 min. In the end, I fill the holls with modelling clay and removed the tube to 37C. It polymerized in 1.5h.
Hope I helped.

Kristin_Engberg

Removing oxygen is definitely important to consistently polymerizing your hydrogel. This can be done by flushing in nitrogen after vacuuming out all the oxygen, as described in the protocol, or by doing an extended flush of nitrogen into the container, as described by shlomilazar.

Andy_Donaldson

When I polymerize, I use a desiccation chamber that is hooked up to a vacuum and an N2 tank. I degass the tube and then I fill the chamber with N2. I've never had a problem with polymerization and It's usually solid at around 2 hours, but I let it go for 3 anyway. I'd say N2 is an important step.

Suzschindler

After I perfuse the mouse, I put the brain in a 20 mL tube that is completely filled with the PFA/acrylamide/bis-acrylamide-activator so there is little to no air in the tube. I store it at 4C for 3+ days, then when I set this at 37C without doing ANY de-gassing procedure, the gel is always polymerized within 3 hours. There are bubbles in the gel, although I haven't noticed any in the brain. Are there any problems with doing it this way?

KeithSiew

@Suzchindler I might try this your way myself and see if I encounter any problems. Let me know if you encounter any issues down the road yourself.

ClarityNL

I used a vacuum desiccator hooked to N2, then vacuum, then again N2, and the gel is polymerizing nicely, no bubbels present. However, when I am degassing and vacuuming, the hydrogel solution is filled with bubbels.. is this normal? The endresult contains no bubbels..

rchurt

The first time I tried to run a brain, after post-fixing it for three days at 40C, the gel didn't polymerize, even though I had degassed it according to the protocol detailed in the paper. Then I tried a tube that I had just taken out of the freezer, and it worked perfectly. I realized that I hadn't been twisting the caps on the 50 mL conical tubes hard enough to completely close them--on the purple-topped VWR conical tubes that I'm using, I learned that I have to turn the cap until I feel it click into place, because otherwise some of the solutes will evaporate during the three-day post-fix.

Try running a fresh tube without a brain in it to see if the post-fixing period is affecting your results.

DavidLenzi

shlomilazar said:

Dear Liz,
Please try the following setup: I fill the 50ml tube (with the hydrogel and brain) with N2 using a syring niddle that is connected to N2 container. I also puctured the lid nearby to let N2 flow to get out. I kept it running for 30 min. In the end, I fill the holls with modelling clay and removed the tube to 37C. It polymerized in 1.5h.
Hope I helped.

I used a similar approach, but found one could replace the air in the 50ml tube with nitrogen in about a minute. Here's how I did it: Punched two 16g needles through the cap of a 50 ml tube, sealed with 5 mn epoxy. On the inside, I added a 5 cm length of tubing to the needle. This is the inflow port to get incoming N2 to the bottom of the tube. On the top, I added on/off Luer valves to the syringes. I next opened both valves, and connected the inflow port to an N2 cylinder at ~2 psi. I let the N2 flow for about 1 minute, then turned off the outflow then the inflow valves. The 50 ml tube went into a 37C water bath. The samples polymerized in about an hour, and there was a bit of positive pressure still evident when I opened the valves indicating the 50ml tube had remained pressurized with N2.

coweight

Dear forum members, gel polymerization does not work for me, and I cannot figure out the reason. Here is what I did:
Basically, I followed the "PACT Clearing" method in this paper "http://www.cell.com/cell/fulltext/S0092-8674(14)00931-3".
1. fix tissue in 4% paraformaldehyde (PFA) first
2. incubate tissue at 4C overnight in hydrogel monomer solution (4% acrylamide, 0.25% photoinitiator 2,2′-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, in 1x PBS).
3. transfer tissue in a glass vial, and put this glass vial in a vacuum desiccator. Fill the desiccator with N2 for 30 second, then vacuum for 10min, finally, turn on N2 again for 30 second, meanwhile, tightly cap the glass vial in the desiccator.
4. transfer the glass vial with tissue in 37C for 3hr. NO POLYMERIZATION! even overnight incubation, NO polymerization.

Could anyone help me out? Thank you!

Adam

There are two possible reasons:
1) You don't have any bis acrylamide in your hydrogel, so it may polymerise within your tissue, but not outside it. You may still find that the clearing process works fine.
2) If the excess hydrogel still doesn't polymerise, then check the pH of your PFA, I've found that it has to be about 7.5 or the gel wont polymerise.

coweight

Adam said:

There are two possible reasons:
1) You don't have any bis acrylamide in your hydrogel, so it may polymerise within your tissue, but not outside it. You may still find that the clearing process works fine.
2) If the excess hydrogel still doesn't polymerise, then check the pH of your PFA, I've found that it has to be about 7.5 or the gel wont polymerise.

Thank you, Adam!
1) When I take the tissue out from the hydrogel monomer solution after embedding for 8 hr, it's still soft, I doubt whether it polymerizes inside. However, I will go on the clearing process.
2) I checked the pH of PFA, it's 6.5, and my hydrogel monomer solution is 7.0 Can I adjust the pH of PFA to 7.5?

Adam

When I tried low density hydrogels, I didn't notice any difference in the brains, but they cleared properly (and very quickly). I would try clearing anyway.
I only had a problem with high pH, not sure if 6.5 would prevent polymerisation. If you have bis-acrylamide, you could always make some hydrogel as per the original clarity protocol and polymerise it. An eppendorf full should polymerise in a few minutes.

coweight

@Adam‌
Thank you!

The first paper talking about clearing method used bis-acrylamide, while the 2nd paper gave an easier method which used acrylamide for this step (2014, cell, "http://www.cell.com/cell/fulltext/S0092-8674(14)00931-3") .

Do both of the protocols work for you?

Adam

They both work, as the hydrogel density is lowered, the clearing speed is increased, at the expense of tissue rigidity (and presumably also a increase in protein loss).