Products

Mounting Medium (Fluoro-KEEPER Antifade)

Product image

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
Image

Antigen retrieval: HistoVT One(#06380)
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 Fragmen(Biotium #20013)

MC3T3-E1 Cell
Image

Blocking: Blocking One Histo(#06349)
1st Ab: Anti-Vimentin Rabbit Polyclonal Antibod (Santa Cruz #sc-7557R)
2nd Ab: CyR2 Goat Anti-Rabbit IgG(H+L)(GENETEX #GTX26940)

MC3T3-E1 Cell
Image

Blocking: Blocking One Histo(#06349)
1st Ab: Anti-Vimentin Rabbit Polyclonal Antibod (Santa Cruz #sc-7557R)
2nd Ab: Cy®2 Goat Anti-Rabbit IgG(H+L)(GENETEX #GTX26940)

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.

Application

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

Application

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.

graf

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.

Application

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

  1. Remove excess liquid from a slide and dispense a couple of drops of the product onto a specimen.
  2. Cover the specimen with a coverslip.
  3. The slide can be observed immediately.
  4. 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

  1. Because the product contains glycerol, it is not recommended for the detection of phycobiliproteins such as phycoerythrin and alloophycocyanin.
  2. Refractive index of Fluoro-KEEPER Antifade Reagent with DAPI and without DAPI is 1.43 - 1.44(20 °C)

Reference

Flouro-KEEPER

  1. H Shoji et al.: Scientific Reports6 (2016)

4% - Paraformaldehyde Phosphate Buffer Solution

  1. K Niikura et al.: Journal of biochemistry159.1 (2016)
  2. T Takenouchi et al.: Immunology letters 167.2 (2015)
  3. K Hirashima et al.: Nucleic acids research 43.4 (2015)
  4. T Takenouchi et al.: Cell biology international 36.12 (2012)
  5. K Onuma et al.: Clinical Orthopaedics and Related ResearchR470.10 (2012)
  6. Y Yamamoto et al.: Thrombosis research 125.3 (2010)

Downloads

Fluoro-KEEPER Antifade Reagent, Non-Hardening Type pdf (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 e-Nacalai
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 e-Nacalai
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 e-Nacalai
Antigen Retrieval
HistoVT One(10x, pH 7.0) SP RT 500 ml 06380-05 e-Nacalai

(Storage) RT: Room temperature, A: Cool and dark, R: Refrigerator, F: Freeze