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Dear list,
we have been imaging BABB cleared samples a few mm thick with great success, but have a problem with one particular sample type, chondrogenic differentiated organoids. The clearing is fine, and staining with Hoechst works well, with nuclei visible throughout the entire volume.
The problem is antibody staining. This is restricted to the first 100 or so microns at the surface of the organoid. Does anyone know of a way to do wholemount staining on dense / difficult to permeabilise cartilage containing samples?
Our current procedure is 4% PFA fixation (overnight).
Block: PBS + 2% TX-100 + 10% FCS + 3% BSA 4h room temp.
Ab incubations in Block buffer plus Ab, 4 days in cold room with rotation. Washes, repeat with secondary Abs, also 4 days.
I am guessing the problem is penetration of the relatively large antibodies into the sample, as the Hoechst work fine.
cheers
Dale
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