I would strongly suggest to use BA/BB mixture for clearing.
Here is the protocol for embryos but can be applied to lung as well I
Methyl salicilate may not be good with 2-photon confocal and may not
be suitbale for some pairs of fluorophores and is nasty with bad smell
Clearing of tissues for microscopy
with benzyl benzoate/benzyl alcohol (BA/BB)
Benzyl benzoate (phenyl methyl benzoate)
(refractive index 1.5685 to 1.570; mixable with alcohol, chloroform,
ether and xylene).
BA/BB solution contains one volume of BA and two volumes of BB (BA: BB = 1:2)
*Benzyl benzoate is intolerant of water! Keep it dessicated!!!
*Use glass lab ware; BA/BB dissolves plastic!!!
*Keep all reagents in the dark!!!
Dehydrate stained embryos (DAB or fluorescence staining) through
ethanol series (70%, 95% and 100% ethanol). Transfer samples into 1:1
mixture of 100% ethanol:BA/BB until the embryos sink (for about 15
min, if sample is mounted on a slide/coverslip). Transfer samples in
BA/BB and incubate until the sample becomes transparent.
Perform imaging in BA/BB.
Reverse the solvent series and store samples in PBS at 40C degrees in the dark.
*BA/BB could gradually dissolve color products of alkaline phosphatase
and B-galactosidase reaction and also fluorophores). Do not keep them
long it BA/BB.
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