perfusion

scottyler89

I just tried the clearing on my first sample, which was pancreas from an animal that someone else was already sacrificing, but their sample meant that I couldn't do the PBS and hydrogel perfusion. Everything seemed to go smoothly regardless though up until the end of my second day clearing, when a vein or artery that had had coagulated blood in it still literally exploded, releasing iron into the media, turning it yellow, likely from iron oxide, which then plated onto the negative electrode. Just a heads up for anyone else who was straying from the protocol.

scottyler89

I also just found a bit of metal in my water bath that seems to have rusted - the more likely culprit.

Kristin_Engberg

Just to comment on performing CLARITY on samples that have not been perfused - it is recommended that you let your sample incubate in the hydrogel solution at 4 degC for a week before embedding. Without perfusion, passive diffusion is the only method for the hydrogel monomers to enter into the sample, and longer incubation times aid in monomer penetration throughout the sample.

joelrosiene

In my experience the sample really does attract all the contaminants in your system. Running your setup for a day or two with any type of contaminant in circulation will generally result in that contaminant being deposited on the surface of your gel. Run your chambers for a day beforehand before adding a sample to them; especially if they are complex.

scottyler89

Kristin_Engberg said:

Just to comment on performing CLARITY on samples that have not been perfused - it is recommended that you let your sample incubate in the hydrogel solution at 4 degC for a week before embedding.

Agreed - that was what I had done, and got very good results for the embedding.

peterlococo

Has anyone found that inclusion of saponin decreases incubation time in non-perfused tissue??