Ethyl Cinnamate clearing troubleshooting

[Ka Ka Yu]

Hi TooT, 

I am troubleshooting my Ethyl Cinnamate clearing for my organoids. My organoids are looking quite cloudy and I can't see the DAPI staining. I have just performed blocking using same blocking buffer as in your paper, stained for DAPI 1:200. For the ethanol dehydration part, I used 30mins per ETOH dehydration step, starting from ETOH diluted in PBS 30%, 50%, 70%, 90%, 100%, 100% again and overnight ETOH dehydration in 100% ETOH. I adjust pH of the ETOH using NaOH and HCl. I previously have used 15mins per ETOH step but it still led to cloudiness. The next day after overnight dehydration, I would aspirate all the ETOH and add ethyl cinnamate at room temperature, allowing the organoids to sit in that for at least 20mins. 

Does anyone have experience troubleshooting this? Thanks so much! 

Best regards,

Ka Ka 

[Abdullah Khan]

Hey Ka Ka, 

It sounds like you're doing everything right - but as you say the image looks quite strange. When you add the ethyl cinnamate, can you see the sample become translucent? 

Is the cloudiness only visible down the microscope? What system are you using to image and do you have any other markers to bench mark? 

Abs