Clearing Pancreas Tissue Using ETC

patiprat

Hello All
I am interested in knowing if anyone in this group was able to clear pancreas tissue using clarity setup, if so what were the conditions you used for the clearing step. I have been clearing two mice pancreas at 25 V 37 degrees for 72 hrs now but I see only partial clearing in my tissue.

Here is the link to the pictures:

https://plus.google.com/photos/102653309454259671450/albums/5923204360943835793?authkey=CI3M8quwoM-FFg

I am continuing to clear the tissue after 72 hrs. I would like to know if you have had any success with clearing pancreas and if you could share insights on clearing this tissue type it would be very helpful. Thank you all for contributing to this forum thus far, I have found the threads in this forum very useful.

nicolasrenier

I don't know how the pancreas will be cleared with Clarity, but you should know that the tissue is not supposed to be completely transparent after the ETC. Your sample looks actually almost good enough at the 4 or 6h time points.

The real clearing is done in the mounting medium. You should stop the ETC before the tissue turns yellow (6h in your case). Once you'll but the pancreas in focusclear, you should have a great transparency. Also, since the tissue seems to clear pretty fast, you should try a 'passive' clearing, in the SDS solution at 37°c for a couple of days and see how it goes. The final aspect after ETC should be 'watery' but not yellow.

patiprat

Thank you very much for your inputs Nicholas. I have been running the passive clearing of the pancreas in parallel to the ETC.
As you mentioned Nicholas the pancreas tissue has become watery and is still colorless, however it is still a cloudy at places. Here are these pictures from the passive clearing. Please let me know whats your take on them.

https://plus.google.com/photos/102653309454259671450/albums/5923209972645744817?authkey=CKzh88zAwf3VrgE

Shashank20

Hi @patiprat, have you tried to stain the ETC pancreas tissue and imaged ? Were you successful in getting staining ?

Shashank20

Hi all,
I got a huge autofluorescence background both in red and green channel while imaging ETC cleared, and Ab-stained mouse pancreas. Unfortunatley I could not see any specific staining. Has anybody experienced similar problem with any other mouse tissue ? Pl. share your experience or how to overcome that ?

LauraLynch

Passive clearing greatly reduces autofluorescence in our brain tissue. Also, don't run ETC at high voltage, high temperature, nor for more than a day or so.

Shashank20

@LauraLynch thanks for your inputs :-)

Clara

Hi, I was trying to stain passive cleared pancreas using anti-insulin antibody but I haven't got a good staining. Anyone working staining pancreas that can share antibodies brands and dilutions? I was using anti-insulin from SantaCruz (sc-9168) 1/50 dilution in PBST, I've tried 4°c and 37°C incubation temperatures for 36h in 1x1mm tissue size.
Thanks in advance for your comments

Leo

@Clara Hi, 1/50 dilution is not important. SantaCruz one has relatively low concentration. I saw my friend who use 1:1 dilution or use original one (not dilution) with SantaCruz one.

Jake

Here is an example of a mouse pancreas cleared by a commercial device: http://logosbio.com/x_clarity/x_clarity/image_gallery.php#pro_taps

jld0072

I'm really late to this party but,
Our lab has been working with clearing whole mouse pancreata and we have had some success using the PACT method with a protocol I've written (a truncated version of the Gradinaru lab's paper). We are able to clear entire an entire pancreas within 1-2 days, without the use of ETC, using an 8% SDS solution in 1x PBS (pH 8.5). We use 4% acrylamide solution for our hydrogel embedding and polymerize for four hours at 37C. Using this method, we have been able to get different antibodies to work (list is growing, albeit slower than I'd like) including insulin.

The antibody we have been using for insulin is the Dako GP anti-insulin antibody. I've dug around a bit, trying to find another good insulin antibody and this one is the gold standard amongst the Pancreas community. I've tried others on 2D sections and none work nearly as well as the Dako Polyclonal Guinea Pig Anti-Insulin A0564 antibody. Using this antibody as our positive control, it should work in just about any condition you're working with when doing CLARITY processing.

So far we've gotten Pdx1, glucagon, insulin, macrophage markers, vasculature markers and neuronal markers to work. We are still early in the process but we are continuing to get this protocol to work with different antibodies against different targets.