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Antifade Reagent: Fluoro-KEEPER, Non-hardening Type

Mounting Medium for Fluorescent Staining / Antifade Reagent

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.

  • Inhibits photobleaching of various fluorescent dyes
  • Easy to use with eye-drop bottle
  • Available with or without DAPI

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
CF™ 488 93 82 91 82
Cy® 2 99 83 98 81
Rhodamine 72 51 78 41
Alexa Fluor® 555 98 81 97 87
CF™ 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

Fluoro-KEEPER (with DAPI)

  • Fluorescent microscopy experiments

    Fluoro-KEEPER with DAPI offers nuclear staining along with mounting.

    Mouse Small Intestine

    Antigen retrieval:  HistoVT One (Cat. No.: 06380)
    Blocking: Blocking One Histo (Cat. No.: 06349)
    1st Ab: Anti-Vimentin Rabbit Polyclonal Antibody
    (Santa Cruz, Cat. No.: sc-7557R)
    2nd Ab: CF™ 488A Goat Anti-Rabbit IgG (H+L), F(ab')2 Fragment
    (Biotium, Cat. No.: 20013)

    MC3T3-E1 Cell

    Blocking:  Blocking One Histo (Cat. No.: 06349)
    Rhodamine-conjugated phalloidin (Cytoskeleton, Cat. No.: HDR1)

    MC3T3-E1 Cell

    Blocking:  Blocking One Histo (Cat. No.: 06349)
    1st Ab: Anti-Vimentin Rabbit Polyclonal Antibody
    (Santa Cruz, Cat. No.: sc-7557R)
    2nd Ab: Cy2® Goat Anti-Rabbit IgG (H+L)
    (GENETEX, Cat. No.: GTX26940)
    Mounting Medium:  Fluoro-KEEPER with DAPI for 30 min. at room temperature protecting from light.
    Microscopy: Olympus BX-50-34-FLA1 
  • Fluorescent microscopy observation in three months

    The samples mounted in Fluoro-KEEPER Antifade Reagent with DAPI can offer brightly signals of DAPI and CF™ 488 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, (Cat. No.: 09154), for 15 min. at RT)
    Blocking:  Blocking One Histo (Cat. No.: 06349)
    1st Ab: Anti-Vimentin Rabbit Polyclonal Antibody (Santa Cruz, Cat. No.: sc-7557R)
    2nd Ab: CF™ 488A Goat Anti- Rabbit IgG (H+L), F(ab') 2 Fragment (Biotium, Cat. No.: 20013)

Fluoro-KEEPER (without DAPI)

  • Fluorescent microscopy experiments (Planarian)

    Nuclear Hoechst 33342
    Arrestin and G Protein β Subunit(Gβ) 1st Ab: Mouse Anti-planarian Arrestin
    1st Ab: 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 

    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.

    Samples were mounted in the Fluoro-KEEPER Antifade Reagent
    Data courtesy of Agata Lab, Department of Biophysics, Kyoto University

    Fluorescent microscopy observation in three months

    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, (Cat. No.: 09154), for 15 min. at RT )
    Retrieval: Histo VT One (Cat. No.: 06380) for 30 min. at 90°C
    Blocking: Blocking One Histo (Cat. No.: 06349)
    1st Ab: Anti-vimentin Rabbit Polyclonal Antibody (Santa Cruz, Cat. No.: sc-7557R)
    2nd Ab: CF™ 488A Goat Anti- rabbit IgG(H+L), F(ab')2 Fragment (Biotium, Cat. No.: 20013)
    CF™ 555 Goat Anti- rabbit IgG(H+L)(Biotium, Cat. No.: 20033)
    Nuclear Staining:  DAPI (Cat. No.: 11034) or Hoechst33258 (Cat. No.: 04907)

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

  • 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)

Ordering Information

Product Cat.No. Storage Quantity Price(US$)  
Fluoro-KEEPER Antifade Reagent, Non-Hardening Type 12593-64 4°C 2 x 5 ml 120.00 Buy
Fluoro-KEEPER Antifade Reagent, Non-Hardening Type with DAPI 12745-74 4°C 2 x 5 ml 135.00 Buy