Mounting Medium (Fluoro-KEEPER Antifade)
Fluoro-KEEPER Antifade Reagent is a non-hardening type mounting medium with a unique antifade reagent. It suppresses rapid photobleaching during fluorescence microscopy experiments. The coverslipped slide with nail polish or other sealants can be stable for several weeks. There are two types of products available, with DAPI [4',6-Diamidino-2- phenylindole] and without DAPI, which counterstains nucleus blue.
Features
- Inhibits photobleaching of various fluorescent dyes
- Easy to use with eye-drop bottle
- Available with or without DAPI
Product Information
Comparison of antifade effectiveness with different fluorescent dyes
Fluoro-KEEPER Antifade Reagent offers increased resistance to photobleaching of various fluorescent dyes.
| without DAPI | with DAPI | |||
| Fluorescence Dye | Fluoro-KEEPER | Control | Fluoro-KEEPER | Control |
| Hoechst 33258 | 98 | 96 | － | － |
| Hoechst 33342 | 100 | 90 | － | － |
| DAPI | 99 | 93 | － | － |
| Propidium Iodide | 95 | 67 | － | － |
| Fluorescein | 97 | 49 | 96 | 49 |
| Alexa Fluor® 488 | 93 | 86 | 96 | 91 |
| CFTM 488 | 93 | 82 | 91 | 82 |
| Cy® 2 | 99 | 83 | 98 | 81 |
| Rhodamine | 72 | 51 | 78 | 41 |
| Alexa Fluor® 555 | 98 | 81 | 97 | 87 |
| CFTM 555 | 98 | 85 | 97 | 85 |
| Cy® 3 | 89 | 71 | 86 | 66 |
Cells stained by each fluorescent dye were mounted in Fluoro-KEEPER Antifade Reagent, 85% Glycerol containing PBS as a control. Samples were illuminated for 60 seconds. Each number indicates fluorescence intensity as percentage of initial intensity after 60 seconds exposure photobleaching.
| Control Condition | 85% Glycerol-PBS (without DAPI), 85% Glycerol-PBS with DAPI (with DAPI) |
|---|---|
| Fluorescent Microscopy | Olympus BX-50-34-FLA1 |
| Exposure time | 60 seconds |
Application
Application 1: Fluorescent Microscopy experiments (Fluoro-KEEPER (with DAPI))
Fluoro-KEEPER with DAPI offers nuclear staining along with mounting.
| Mounting Medium | Fluoro-KEEPER with DAPI for 30 min. at room temperature protecting from light. |
|---|---|
| Microscopy | Olympus BX-50-34-FLA1 |
| Mouse Small Intestine | |
|---|---|
 |
Antigen retrieval: HistoVT One（#06380） |
| MC3T3-E1 Cell | |
 |
Blocking： Blocking One Histo（#06349） |
| MC3T3-E1 Cell | |
 |
Blocking： Blocking One Histo（#06349） |
Application 2: Fluorescent microscopy observation in three months (Fluoro-KEEPER (with DAPI))
The samples mounted in Fluoro-KEEPER Antifade Reagent with DAPI can offer brightly signals of DAPI and CF488TM in spite of storage for three months upon sealing of a coverslip. For long-term storage, seal with hot wax or nail polish and store at 4 °C protecting from light. Please refer to the instruction for more details.
| Sample | MC3T3-E1 cell (Fixation: 4%-Paraformaldehyde Phosphate Buffer Solution, (#09154), for 15 min. at RT) |
|---|---|
| Blocking | Blocking One Histo (#06349) |
| 1st Ab | Anti-Vimentin Rabbit Polyclonal Antibody (Santa Cruz #sc-7557R) |
| 2nd Ab | CFTM 488A Goat Anti-Rabbit IgG (H+L), F(ab’)2 Fragment (Biotium #20013) |
Application 3: Fluoro-KEEPER (without DAPI)
Fluorescent microscopy experiments; Planarian
Fluorescence intensity
Fluorescence intensities are shown as percentages of initial intensities remaining during repeated frame capture up to 60 times. The images were acquired by Olympus FV10. The samples mounted in the Fluoro-KEEPER Antifade Reagent were clearly detected after 60 times of frame capture.
| Nuclear | Hoechst 33342 |
|---|---|
| Arrestin and G Protein β Subunit(Gβ) | 1st Ab: Mouse Anti-planarian Arrestin Mouse Anti-planarian Gβ 2nd Ab: Alexa Fluor® 488 Goat Anti-mouse IgG |
| Tryptophan β Hydroxylase(TPβH) | 1st Ab: Rabbit Anti-planarian TPβH 2nd Ab: Alexa Fluor® 594 Goat Anti-rabbit IgG |
Samples were mounted in the Fluoro-KEEPER Antifade Reagent
Data courtesy of Agata Lab, Department of Biophysics, Kyoto University
Application 4: Fluoro-KEEPER (without DAPI)
Fluorescent microscopy observation
The samples mounted in Fluoro-KEEPER Antifade Reagent can be stored for long-term when the coverslip are completely sealed with nail polish.
| Sealing | Nail polish |
|---|---|
| Storage | 3 months at 4 °C protected from light |
| Sample | MC3T3-E1 cell (Fixation: 4%-Paraformaldehyde Phosphate Buffer Solution, (#09154), for 15 min. at RT ) |
| Retrieval | Histo VT One (#06380) for 30 min. at 90°C |
| Blocking | Blocking One Histo (#06349) |
| 1st Ab | Anti-vimentin Rabbit Polyclonal Antibody (Santa Cruz #sc-7557R) |
| 2nd Ab | CF™ 488A Goat Anti-rabbit IgG(H+L), F(ab’)2 Fragment (Biotium #20013) CF? 555 Goat Anti-rabbit IgG(H+L)(Biotium #20033) |
| Nuclear Staining | DAPI (#11034) or Hoechst33258 (#04907) |
Protocols
Protocol
- Remove excess liquid from a slide and dispense a couple of drops of the product onto a specimen.
- Cover the specimen with a coverslip.
- The slide can be observed immediately.
- To obtain sufficient counterstain, incubate the slide for 30 min. at room temperature protecting from light.
For long-term storage, sealing with hot wax or nail polish and store at 4 °C protected from light.
Precaution
- Because the product contains glycerol, it is not recommended for the detection of phycobiliproteins such as phycoerythrin and alloophycocyanin.
- Refractive index of Fluoro-KEEPER Antifade Reagent with DAPI and without DAPI is 1.43 - 1.44(20 °C)
Reference
Flouro-KEEPER
- H Shoji et al.: Scientific Reports6 (2016)
4% - Paraformaldehyde Phosphate Buffer Solution
- K Niikura et al.: Journal of biochemistry159.1 (2016)
- T Takenouchi et al.: Immunology letters 167.2 (2015)
- K Hirashima et al.: Nucleic acids research 43.4 (2015)
- T Takenouchi et al.: Cell biology international 36.12 (2012)
- K Onuma et al.: Clinical Orthopaedics and Related ResearchR470.10 (2012)
- Y Yamamoto et al.: Thrombosis research 125.3 (2010)
Downloads
Fluoro-KEEPER Antifade Reagent, Non-Hardening Type 
(PDF 3.3MB)
Ordering Information
| Product Name | Grade | Storage | PKG Size | Product No. | Price |
|---|---|---|---|---|---|
| Fluoro-KEEPER Antifade Reagent, Non-Hardening Type | SP | R | 2 x 5 ml | 12593-64 |  |
| Fluoro-KEEPER Antifade Reagent, Non-Hardening Type with DAPI | SP | R | 2 x 5 ml | 12745-74 |
Related Products
| Product Name | Grade | Storage | PKG Size | Product No. | Price |
|---|---|---|---|---|---|
| Fixation | |||||
| 10% - Formaldehyde Neutral Buffer Solution | - | RT | 1 L | 37152-51 | |
| 4% - Paraformaldehyde Phosphate Buffer Solution | SP | R | 5 x 10 ml | 09154-14 | |
| 09154-85 | |||||
| Blocking Reagents | |||||
| Blocking One Histo | SP | R | 50 ml | 06349-64 | |
| Antigen Retrieval | |||||
| HistoVT One(10x, pH 7.0) | SP | RT | 500 ml | 06380-05 | |
(Storage) RT: Room temperature, A: Cool and dark, R: Refrigerator, F: Freeze
