Tissue clearing was performed as described by Kurihara et al. (2015) (link) with slightly modifications. ClearSee solutions were prepared by mixing xylitol powder [10% (w/v) final concentration; Wako, Osaka, Japan, 248-00545], sodium deoxycholate [15% (w/v) final concentration; Tokyo Chemical Industry, Tokyo, Japan, C0316] and urea [25% (w/v) final concentration; Wako, 211-01213] in water. The following reductants (50 mM final concentration) were used for screening: reduced glutathione (Wako, 071-02014); 1-thioglycerol (Nacalai Tesque, Kyoto, Japan, 33709-62); 2-mercaptoethanol (Wako, 131-14572); 1,4-dithiothreitol (Wako, 048-29224); 2-aminoethanethiol hydrochloride (Nacalai Tesque, 21419-32); and sodium sulfite (Wako, 190-03411). The leaves were fixed in 4% (w/v) PFA (Wako, 162-16065) for 120 min in PBS under vacuum (690 mmHg) at room temperature. Fixed tissues were washed twice for 1 min in PBS and cleared with modified ClearSee at room temperature or 4°C until clearing.
Protocol detail
Tissue Clearing and Reductive Screening
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2 aminoethanethiol hydrochloride
2 mercaptoethanol
Clearsee
Dithiothreitol
Powder
Reduced glutathione
Reductants
Sodium deoxycholate
Sodium sulfite
Thioglycerol
Tissues
Urea
Vacuum
Xylitol
Corresponding Organization : Nagoya University
Other organizations : The University of Tokyo
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Variable analysis
independent variables
- Reductants (reduced glutathione, 1-thioglycerol, 2-mercaptoethanol, 1,4-dithiothreitol, 2-aminoethanethiol hydrochloride, sodium sulfite) used for screening at 50 mM final concentration
- Temperature (room temperature or 4°C) used for tissue clearing
- Tissue clearing efficiency
- Tissue fixation in 4% (w/v) PFA for 120 min in PBS under vacuum (690 mmHg) at room temperature
- Tissue washing in PBS twice for 1 min
- Modified ClearSee solution composition (xylitol 10% (w/v), sodium deoxycholate 15% (w/v), urea 25% (w/v))
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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