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HistoBrightTM <Tissue Structure Fluorescent Dye>
Catalog NO. FDV-0051
Research use only, not for human or animal therapeutic or diagnostic use.
This product has been commercialized under the license from Kochi University and Ehime University.

Product Background
Tissue structures are historically visualized by staining with various colorimetric dyes such as Hematoxylin
and Eosin (HE) stains. These conventional colorimetric stains generally require thinly sliced tissue sections and areunsuitable for thick tissue blocks. However, the preparation of thinly sliced tissues loses the 3D structural information.
To reconstitute 3D information, a lot of additional thin slices and stainings are required. HistoBrightTM (originalcompound name; PC in Ref.1) is originally synthesized by Dr. Yosuke Niko, Kochi University, and Dr. Takeshi
Imamura, Dr. Masamoto Murakami, and Dr. Ryosuke Kawakami, Ehime University, promotes its biological 3D
imaging application. HistoBrightTM is a novel solvatochromic fluorescent dye that changes fluorescent colors inresponse to solvents or microenvironments and has a high affinity to various biological membranes. The combinationof two features of HistoBrightTM contributes to clear and bright imaging of tissue structures.
As HistoBrightTM can be excited by not only one-photon but also two-photon, HistoBrightTM is compatible
with two-photon microscopy and useful for tissue imaging. Furthermore, HistoBrightTM, coupled with several tissueclearing methods such as LUCID and RapiClear, which do not require lipid removal, contributes to analyzing 3D
deep tissue imaging of thick tissue slices or blocks, etc., without breaking pathophysiological tissue structures.
HistoBrightTM is an innovative fluorescent dye for the visualization of 3D tissue structures.

Figure 1 Overview of HistoBrightTM stainingDescription
Catalog Number: FDV-0051
Size: 0.1 mg
Formulation: C27H27NO
Molecular weight: 381.5 g/mol
Solubility: Soluble in DMSO
Fluorescent characteristics:
Ex. 350-500 nm (maximum ~410 nm)
Compatible with 405-488 nm laser (confocal microscopy), 960-1100 nm laser (two-photon microscopy)
Em. 500-800 nm (depending on solvents)

Reconstitution and Storage
Reconstitution: Stock solution recommended concentration 1 mM in 100% DMSO.
Storage (powder): Store powder at -20°C.
Storage (solution): After reconstitution in DMSO, aliquot and store at -20°C.
Avoid repeated freeze-thaw cycles.

How to use
General procedure for tissue staining*This procedure is an example of HistoBrightTM
1. Tissue samples were excised and then fixed in freshly prepared paraformaldehyde.
2. Tissue blocks or thick slices prepared by any appropriate methods.
NOTE: As HistoBrightTM is a membrane staining dye, any de-lipid procedures including organic solvents or highconcetration of detergents such as Triton X100, Tween 20 etc. do not recommened. If you need to use
detergents for permealization etc., please optimize experimental condition.
3. Stock solution of HistoBrightTM (1 mM) was mixed in tissue clearing solution (LUCID or RapiClear etc.) toprepare the staining and clearing solution (hereafter called Master mix). The initial recommendation ofHistoBrightTM concentration is 10 M.
4. Immerse tissue blocks/sections in the Master mix for over 48 hours at room temperature.
5. After staining tissue blocks/sections, image by confocal laser microscopy or two-photon microscopy.
NOTE: Empirically optimize staining conditions (tissue sample size, pre-treatment of tissues, the concentrationof HistoBrightTM, clearing methods, staining time, etc.) for your experiments.

Reference data
Solvatochromic property of HistoBrightTM
Absorbance and fluorescent spectrum of HistoBrightTM
in various solvents. While the absorbance spectrum ofHistoBrightTM is not sensitive to solvent polarity,
HistoBrightTM changes fluorescent color from green to
far-red depending on solvent polarity. Under a lowpolarity environment, HistoBrightTM emits green color.
On the other hand, under a high polarity environment,
HistoBrightTM emits red to far-red with low quantum
yield.
Quantum yields are 0.82 (Toluene), 0.77 (EtOAc), 0.83
(CHCl3), 0.65 (MeCN), 0.20 (EtOH), and 0.07 (MeOH).
[ver. 2024/05]

Download the latest datasheet from www.funakoshi.co.jp(Japanese)www.funakoshi.co.jp/exports (English)Fluorescent spectrum on a liposome membrane model
HistoBrightTM rapidly accumulates in lipid membranes and emits differentfluorescent colors in response to the membrane environments. On the liquidorder (Lo) model liposome (sphingomyelin (SM)/cholesterol (Chol)),
HistoBrightTM emits a maximum of around 575 nm fluorescence with a highquantum yield value (0.72). On the other hand, HistoBrightTM on the liquid
disorder (Ld) model liposome (DOPC) exhibits significantly broad andrelatively weak fluorescence (quantum yield 0.37), and the maximum
wavelength is red-shifted by approximately 30 nm compared to the Lomodel.

Application data
Two-photon fluorescence microscopy imaging of human normal skin tissue
After fixation of human skin tissue blocks with 4% paraformaldehyde/PBS, 500 m thick slides were prepared andtreated with the LUCID clearing reagent and 10 M HistoBrightTM for 76 hours. The tissue slides were observed by
two-photon fluorescence microscopy (Ex. 960 nm, Em. cyan channel: 492 nm as SHG, green channel: 500-550 nm,
orange channel: 560-593 nm, and red channel: 593-690 nm). The merged image was obtained from four channels.

3D images were reconstructed from Z-stack sequences (step size, 5 m) of 2D images.

Comparison between confocal microscopy and two-photon microscopy
imaging
After fixation of human skin tissue blocks with 4%paraformaldehyde/PBS, 500 m thick slides were prepared and treatedwith the RapiClear clearing reagent and 10 M HistoBrightTM for 72
hours without any permealization treatments. Tissues were observed byconfocal microscopy (488 nm laser) and two-photon microscopy (960nm laser).

HE staining after HistoBrightTM fluorescent imaging
After fluorescent imaging, the stained tissue block waswashed with PBS. Thin tissue slices were prepared from thetissue block and re-stained with the conventional HE
staining procedure.

Notes
All spectrum data were obtained by Dr. Yosuke Niko, Kochi University.
All cell imaging data were obtained by Dr. Ryosuke Kawakami, Ehime University.

Reference
1. Inoue et al.,J. Mater. Chem. B, 10, 1641-1649 (2022) Synthesis and photophysical properties of a new push-pull
pyrene dye with green-to-far-red emission and its application to human cellular and skin tissue imaging.
Disclaimer/免責事項
This product has been commercialized by Funakoshi Co., Ltd.
based on the results of academic research, and the advertisement
text, figures and manuals (hereinafter “Product information”) havebeen prepared based on published research reports on April, 2023.
The academic interpretation at the time of creation of the Product
Information may change in accordance with future developmentsin the relevant research field and expansion of various scientificfindings, and the latest version and certainty of the Product
Information are not guaranteed. The specifications of this productand the Product Information are subject to change without notice.
Please contact us for the latest information.
本商品は学術研究成果を基にフナコシ株式会社が商品化したもので、2023 年 4 月時点における公開研究報告を基に広告文章およびマニュアル（以下、商品資料）を作成しています。今後の当該研究分野の発展および各種学術知見の拡大にともない、商品資料作成時の学術的解釈が変更になる可能性があり、最新性・確実性を保証するものではありません。また、本商品の仕様および商品資料を予告なく変更する場合がございます。最新の情報に関しましては、弊社までご確認いただきますようお願い申し上げます。

[ver. 2024/05]

Download the latest datasheet from www.funakoshi.co.jp(Japanese)www.funakoshi.co.jp/exports (English)