Q: What kind of staining reagents can be used?
A: a) Antibodies: direct fluorescent-labeled antibodies are preferred. For example, the antibodies are diluted adequately in PBS containing both 0.5% Triton™ X-100 and 0.01% NaN3.
b) Nuclei staining reagents: propidium iodide can be used. Propidium iodide is diluted to 10 µg/mL with 0.1 M phosphate buffer (pH 7.4), containing 0.5 M NaCl.
Q: Can antibodies from my laboratory be used?
A: Some proteins do not change their antigenicity during fixation or tissue-clearing procedures. However, this is not conformed for all the proteins. It is recommended to initially test the antibodies you are already using.
Q: Can fluorescent-labeled secondary antibodies be used?
A: We do not have information about protocols using secondary antibodies. It takes significant amounts of time for the two-step treatment with both primary and secondary antibodies. Therefore, we recommend directly labeling your primary antibody with fluorescent reagents.
Q: What kind of fluorescent proteins can be used?
A: We assessed the retention of fluorescence intensity in GFP. Some fluorescent proteins, such as, EGFP, EYFP, mCherry, and mKate2, have been confirmed to retain their fluorescence signals (Cell 2014, 157, 726.)
Q: What kind of fluorescent labeling dyes can be used for immunohistochemical staining during clearing step?