animal tissue-clearing reagent cubic€¦ · tcichemicals.com animal tissue-clearing reagent cubic...
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Animal Tissue-Clearing ReagentCUBIC
Advantages・Basic protocol; Soaking in only two products makes mouse whole-body or animal tissues clear! T3740 CUBIC-L: for delipidation and decoloring T3741 CUBIC-R+: for RI matching・Optional protocol; Optional products can easily clear tissues which were previously difficult to clear. T3780 CUBIC-B: for bone T3781 CUBIC-HL: for highly fatty tissues and quenching autofluorescence T3782 CUBIC-P: for mouse perfusion efficiently aids with perfusion fixation.・Expansion protocol; The following products can clear tissues with expansion. T3866 CUBIC-X1: for expansion tissues T3867 CUBIC-X2: for RI matching with keeping the expanded size・ Preserve the fluorescent protein signals except CUBIC-HL(T3781).・ Shorter sample treatment period.・ Using light-sheet fluorescent microscopy (LSFM) or confocal laser-scanning microscopy (CLSM) enables the whole-organ / body imaging at a cellular resolution.
These products were developed by Prof. Hiroki R. Ueda (The University of Tokyo / RIKEN) and are under invention licenses by RIKEN, Japan.
Whole-body clearing Whole-body clearingwith propidium iodide staining
Tissue-Clearing Reagent CUBIC-L [for Animals] 25mL / 100mL [T3740]Tissue-Clearing Reagent CUBIC-R+ [for Animals] 25mL / 100mL [T3741]Tissue-Clearing Reagent CUBIC-B [for Animals] 25mL / 100mL [T3780]Tissue-Clearing Reagent CUBIC-HL [for Animals] 25mL / 100mL [T3781]Tissue-Clearing Reagent CUBIC-P [for Animals] 25mL / 100mL [T3782]Tissue-Clearing Reagent CUBIC-X1 [for Animals] 25mL / 100mL [T3866]Tissue-Clearing Reagent CUBIC-X2 [for Animals] 25mL / 100mL [T3867]
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Use a tube wherein the whole body can be submerged.Please refer to Q&A about staining reagents.PFA : paraformaldehyde, RT : room temperature
Perfusionfixation
Process Reagent Temp. Time Notes
After perfusion, the mouse needs to be perfused with 50% CUBIC-L (1:1 mixture of water and CUBIC-L : water).
ＰＢＳ4% PFA in PBSＰＢＳ
ＰＢＳ
> 6 hr
> 5 days
> 2 hr x 3> 3 days> 2 hr x 3 Total 1 day, optional
３７℃
３７℃
ＲＴＲＴＲＴ
50% CUBIC-L
Staining reagents
50% CUBIC-L
CUBIC-L
ＰＢＳ
Perfusion
Delipidation
Wash x 3(Staining)(Wash x 3)
(Pre-treatment)Completely immerse the whole body of the mouse with gentle shaking (same in following steps). OptionalRefresh CUBIC-L on day 1, day 2 and every 2 days after day 4.
Optional
1 day> 1 day
ＲＴＲＴ
50% CUBIC-Ｒ+CUBIC-Ｒ+
Pre-treatmentRI matching
1:1 mixture of water and CUBIC-R+
Total 1 day｠
Wash x 3PBS
> 2 hr x 3
(Wash x 3)PBS
> 2 hr x 3
Pre-treatment50% CUBIC-L
> 6 hr
Delipidation
> 5 daysCUBIC-L
（Staining）
> 3 daysStains
Pre-treatment
1 day50% CUBIC-R+
RI match
> 1 dayCUBIC-R+
Whole-body clearing Whole-body clearingwith propidium iodide staining
Direction for Use : Mouse whole-body clearing protocol
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Direction for Use : Mouse whole-organ clearing protocol
Whole-body clearingwith nuclei staining and immunostaining
Whole-brain clearing
Use a tube whose diameter is a slightly larger than that of the organs, and the volume of reagents is half of that of tubes.
Process Reagent Temp. Time Notes
1 day
> 2 hr x 3
4℃
ＲＴ
4% PFA in PBS
PBS
Tissue excisionTissue Fix
Wash x 3 Shake gently (Same in following steps). Total 1 day
6 ‒ 24 hr
> 2 days
37℃
37℃
50% CUBIC-L
CUBIC-LDelipidation
(Pre-treatment) 1:1 mixture of water and CUBIC-L. Optional
Refresh CUBIC-L on day 1, day 2 and every 2 days after day 4.
> 2 hr x 3
> 3 days
ＲＴ
ＲＴ
PBS
Staining reagents
Wash x 3(Staining) Optional
Total 1 day
Total 1 day, optional> 2 hr x 3ＲＴPBS(Wash x 3)
After perfusion fixation
1 dayＲＴ50% CUBIC-R+Pre-treatment 1:1 mixture of water and CUBIC-R+
> 1 dayＲＴCUBIC-R+RI matching
Wash x 3PBS
> 2 hr x 3
(Wash x 3)PBS
> 2 hr x 3
Pre-treatment50% CUBIC-L6 ‒ 24 hr
Delipidation
> 2 daysCUBIC-L
（Staining）
> 3 daysStains
Pre-treatment
1 day50% CUBIC-R+
RI match
> 1 dayCUBIC-R+
Wash x 3PBS
> 2 hr x 3
Fix4％ PFA1 day
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Animal Tissue-Clearing Reagent CUBIC
Direction for Use : For the e�cient clearing of adult mouse(more than 6-week-old) whole-body or organ samples
*If the immersion period is longer than 4 days, the CUBIC-L should be replaced at least once.
(Wash)PBS1 day
PerfusionCUBIC-P
WashPBS1 day
(Staining)Stains
5 ‒ 7 days
Delipidation
3 ‒ 7 days*CUBIC-L
Pre-treatment
1 day50% CUBIC-R+
RI matching
1 ‒ 2 daysCUBIC-R+
Perfusionfixation
Process Reagent Temp. Time Notes
After perfusion of CUBIC-P, the organs are dissected.
15ｍＬ PBS20mL 4% PFA in PBS15ｍＬ PBS
PBS 1 day1 day
1 ‒ 2 days
ＲＴＲＴ
ＲＴ
100mL CUBIC-P
50% CUBIC-Ｒ+
CUBIC-Ｒ+
(Wash)Pre-treatmentRI matching
PentobarbitalSacrifice
CUBIC-L 3 ‒ 7 days*1 day
5 ‒ 7 days
37℃ＲＴ
ＲＴ
PBS
Staining reagents
DelipidationWash(Staining)
Shake gently (same in following steps).
Optional
Optional
1:1 mixture of water and CUBIC-R+
4℃
Overdose of pentobarbital
Process Reagent Temp. Time Notes
3 ‒ 7 days*1 day
1 day37℃4℃
ＲＴCUBIC-L4% PFA in PBS
PBS
Tissue fixationDelipidationWash
5 ‒ 7 days
1 day
ＲＴ
ＲＴ
Staining reagents
PBS
(Staining)(Wash) Optional
Optional
1:1 mixture of water and CUBIC-R+1 dayＲＴ50% CUBIC-R+Pre-treatment
Shake gently (same in following steps).
1 ‒ 2 daysＲＴCUBIC-R+RI matching
5 ‒ 7 days
1 day
37℃
ＲＴ
CUBIC-B
PBS
DecalcificationWash
The CUBIC-B should be refreshed at least once.
2 ‒ 4 days37℃CUBIC-LDelipidation1 dayＲＴPBSWash
*If the immersion period is longer than 4 days, the CUBIC-L should be replaced at least once.
(Wash)PBS1 day
DelipidationCUBIC-Ｌ
DecalcificationCUBIC-B5 ‒ 7 days
(Staining)Stains
5 ‒ 7 days
Wash
1 dayPBS
Wash
1 dayPBS
Wash
1 dayPBS
Pre-treatment
1 day50% CUBIC-R+
RI matching
1 ‒ 2 daysCUBIC-R+
3 ‒ 7 days*
DelipidationCUBIC-Ｌ2 ‒ 4 days
Mouse leg
Clearing examples
Mouse head Mouse body Mouse spine
Direction for Use : For mouse body or tissues including bone
透明化の操作に関する質問
Direction for Use : For large blocks of human brain tissue
The autofluorescence of cerebral cells decreased as the delipidation period increased. In order to preserve sufficient autofluorescent signals, delipidation should be finished within approximately one week.
Process Reagent Temp. Time Notes
1 day4℃ＲＴ
FormalinPBS
FixationWash
Stored until use.｠
Shake gently (same in following steps).
1 ‒ 2 weeks45℃CUBIC-LDelipidation The CUBIC-L should be refreshed at least once.
1 dayＲＴPBSWash1 dayＲＴ50% CUBIC-R+Pre-treatment 1:1 mixture of water and CUBIC-R+
1 ‒ 2 daysＲＴCUBIC-R+RI matching
WashPBS1 day
Delipidation
1 ‒ 2 weeksCUBIC-L
Pre-treatment
1 day50% CUBIC-R+
RI matching
1 ‒ 2 daysCUBIC-R+
WashPBS1 day
Process Reagent Temp. Time Notes
1 day4℃ＲＴ
FormalinPBS
FixationWash Shake gently (same in following steps).
Stored until use.
1 ‒ 2weeks**
37℃or 45℃CUBIC-ＨLDelipidation 37 ℃ for human brain or kidney,
45 ℃ for human heart, liver, lung or spleen
1 day
5 ‒ 7 days
ＲＴ
ＲＴ
PBS
Staining reagents
Wash(Staining) Optional
Optional1 dayＲＴPBS(Wash)1 dayＲＴ50% CUBIC-R+Pre-treatment 1:1 mixture of water and CUBIC-R+
1 ‒ 2 daysＲＴCUBIC-R+RI matching**The immersion period is based on the sample size. As delipidation progresses, the apparent opacity inside the sample disappears. During delipidation, the CUBIC-HL should be replaced at least once. If delipidation needs to be prolonged beyond 2 weeks, the subsequent delipidation is recommended to be done at a lower temperature or in CUBIC-L (T3740).
WashPBS1 day
(Wash)PBS1 day
(Staining)
5 ‒ 7 daysStains
Pre-treatment
1 day50% CUBIC-R+
RI matching
1 ‒ 2 daysCUBIC-R+
DelipidationCUBIC-HL
1 ‒ 2 weeks**
WashPBS1 day
Human-brain clearing
Human heart Human kidney
Clearing examples
Direction for Use : For aggressive human tissue clearing
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ReferencesA three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing Tatsuya C. Murakami, Tomoyuki Mano, Shu Saikawa, Shuhei A. Horiguchi, Daichi Shigeta, Kousuke Baba, Hiroshi Sekiya, Yoshihiro Shimizu, Kenji F. Tanaka, Hiroshi Kiyonari, Masamitsu Iino, Hideki Mochizuki, Kazuki Tainaka and Hiroki R. Ueda, nature neuroscience, 2018, 24, 625-637.
Chemical Landscape for Tissue Clearing based on Hydrophilic Reagents K. Tainaka, T. C. Murakami, E. A. Susaki, C. Shimizu, R. Saito, K. Takahashi, A. Hayashi-Takagi, H. Sekiya, Y. Arima, S. Nojima, M. Ikemura, T. Ushiku, Y. Shimizu, M. Murakami, K. F. Tanaka, M. Iino, H. Kasai, T. Sasaoka, K. Kobayashi, K. Miyazono, E. Morii, T. Isa, M. Fukayama, A. Kakita, H. R. Ueda, Cell Reports 2018, 24, 2196-2210.
Whole-Body Profiling of Cancer Metastasis with Single-Cell Resolution Shimpei I. Kubota, Kei Takahashi, Jun Mishida, Yasuyuki Morishita, Shogo Ehata, Kazuki Tainaka, Kohei Miyazono and Hiroki R. Ueda, Cell Reports 2017, 20, 236-250.
Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis Etsuo A. Susaki, Kazuki Tainaka, Dimitri Perrin, Fumikai Kishino, Takehiro Tawara, Tomonobu M. Watanabe, Chihiro Yokoyama, Hirotaka Onoe, Megumi Eguchi, Shun Yamaguchi, Takaya Abe, Hiroshi Kiyonari, Yoshihiro Shimizu, Atshshi Miyawaki, Hideo Yokota and Hiroki R. Ueda, Cell 2014, 157, 726-739.
Whole-Body Imaging with Single-Cell Resolution by Tissue Decolorization Kazuki Tainaka, Shimpei I. Kubota, Takeru Q. Suyama, Etsuo A. Susaki, Dimitri Perrin, Maki Ukai-Tadenuma, Hideki Ukai and Hiroki R. Ueda, Cell 2014, 159, 911-924.
For further information please refer to our website at www.TCIchemicals.com. ▼ ▼ ▼ tissue clearing
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Direction for Use : Mouse whole-brain clearing with expansion protocol
*For nuclear staining, use 30 μg/mL Propidium iodide (PI) and 1.5 M NaCl in PBS.Since the expanded brains are fragile, careful handling is required after the swelling step.
Process Reagent Temp. Time Notes
1 day
> 2 hr x 3
4℃
ＲＴ
4% PFA in PBS
PBS
Tissue excisionTissue Fix
Wash x 3 Shake gently (Same in following steps). Total 1 day
3 hr
5 ‒ 14days
37℃
37℃
50% CUBIC-L
CUBIC-LDelipidation
Pre-treatment 1:1 mixture of water and CUBIC-L.
Refresh CUBIC-L every 4 days.5 days for 1-week-old mice7 days for 3-week-old mice14 days for 8-week-old and 6-month-old mice
1 day
3 days
ＲＴ
ＲＴ
PBS
Staining reagents*
WashStaining
1 dayＲＴPBSWash
After perfusion fixation
2.5 days4℃CUBIC-X1SwellingRefresh CUBIC-X2 every 12 hours.1.5 daysＲＴCUBIC-X2RI matching
WashPBS1 day
WashPBS1 day
Pre-treatment50% CUBIC-L
3 hr
Delipidation
5 ‒ 14 daysCUBIC-L
Staining
3 daysStains
Swelling
2.5 dayCUBIC-X1
RI match
1.5 dayCUBIC-X2
Wash x 3PBS
> 2 hr x 3
Fix4％ PFA1 day
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