Sodium deoxycholate

The following chemicals were of either an analytical or reagent grade and used without further pretreatment. IR-820 dye (CAS No 172616–80-7) denoted as “IR”, tetramethylrhodamine isothiocyanate-dextran (TRITC-Dex40; MW = 40kDa), phenol (CAS No 108–95-2), 5-bromo-4-chloro-3-indolyl phosphate disodium salt (BCIP; CAS No 38404–93-2), and Triton^® X-100 (CAS No 9002–93-1) were purchased from Sigma-Aldrich Co (Missouri, USA). Soybean lecithin (CAS No 8002–43-5), cholesterol (CAS No 57–88-5), sodium deoxycholate (CAS No 302–95-4), 9,10-dimethylanthracene (DMA; CAS No 781–43-1), and gallic acid hydrate (CAS No 5995–86-8) were obtained from Tokyo Chemical Industry Co, Ltd (Tokyo, Japan). Ammonium ferrous sulfate hexahydrate (CAS No 7783–85-9), butylated hydroxytoluene (BHT; CAS No 128–37-0), and xylenol orange (CAS No 3618–43-7) were obtained from Loba Chemie Pvt Ltd (Mumbai, India). Acetone (CAS No 67–64-1), chloroform (CAS No 67–66-3), and methanol (CAS No 67–56-1) were supplied from VWR International Company (Pennsylvania, USA), RCI Labscan Limited. (Bangkok, Thailand), and Fisher Scientific Co LLC (Pennsylvania, USA), respectively. Type 1 (ultrapure) water was produced by using an arium^® pro ultrapure water system (Sartorius AG, Germany) to maintain the electrical conductivity at 0.055 μS/cm at room temperature and was used for all the experiments.

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.