FocusClear

nicolasrenier

@David : I tried (2) that with an adult brain, and it didn't worked : the tissue seems somewhat clear at the beginning but gets cloudy really fast.

LauraLynch

@jjsun90: This is what we're using, from Sigma:

ß-Nicotinamide adenine dinucleotide phosphate hydrate (NADP) (N5755-100MG)

P_Viskaitis

For me simply 80% glycerol worked fine and did not observe any opacity spread problems

Fabio

What tissue did you clear? or was it just a slice?

achung

I just imaged my first cleared brain in 80% glycerol. While imaging, it worked very well. However, I noticed that after about 90 minutes, a white precipitate started to form on the outside of the brain. I put it back into the clearing solution and after a few hours the precipitate disappeared, but this could result in some issues for imaging substantial/multiple areas.

rspence

given the cost of focus clear what are your thoughts about using more than once? seems like it could be a bad thing since im sure the makeup of the focus clear would change a bit after each use

Shashank20

Has anybody tried using BABB (Murray's Clear- refractive index 1.559) as a mounting medium after deyhrating the ETC run, and antibody stained tissue ??

KatherineHolzem

@LauraLynch... do you know what is the role of the NADPH in the medium?

Solder

so does using gycerol work? Is it a matter of not working as well as focusclear, or has glycerol had any adverse side effects on the tissue?

LauraLynch

@KatherineHolzem: No clue. All is know is that our fake version doesn't work. Details are in the "Loss of GFP Signal" thread. We've had better luck with ScaleU2.

Harris_M

Shashank20 said:

Has anybody tried using BABB (Murray's Clear- refractive index 1.559) as a mounting medium after deyhrating the ETC run, and antibody stained tissue ??

I use BABB routinely but for Optical Projection Tomography (OPT), to get 3D datasets and output. Works well though GFP and it's derivatives don't work as the methanol pre-treatment denatures the protein!!

Hence my reason for starting here to see an alternative method to use with OPT. Saw a non-ETC FocusClear'd E12.5 mouse embryo yesterday, looked quite good and definately better than 80% glycerol.

With the ETC step, does everyone use this and if yes, have to had to make your own chambers, ensuring they're lightproof to stop bleaching of GFP, or similar, labelled samples?

Cheers

Harris

aysegul

P_Viskaitis said:

For me simply 80% glycerol worked fine and did not observe any opacity spread problems

how to prepare %80 glycerol solution without bubbles since it is very viscous liquid? Is there any trich for preparing ?

aysegul

@P_Viskaitis how to prepare %80 glycerol solution without bubbles since it is very viscous liquid? Is there any trich for preparing ?