Fluoro-KEEPER Antifade Reagent offers increased resistance to photobleaching of various fluorescent dyes.

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.

• 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)

• 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)

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)

 Fluoro-KEEPER Antifade Reagent, Non-Hardening Type (Instruction)

 Fluoro-KEEPER Antifade Reagent, Non-Hardening Type with DAPI (Instruction)

• Yin, Xiaobo, et al. "Expression and cell transformation activity of dynactin‐associated protein isoforms." FEBS Open Bio (2021).

• Sugiura, Kenta, and Midori Matsumoto. "Spermatozoa morphology changes during reproduction and first observation of acrosomal contact in two dioecious species of Macrobiotidae (Tardigrada: Eutardigrada)." Zygote 29.1 (2021): 42-48.

• Kobori, Takuro, et al. "Subcellular distribution of ezrin/radixin/moesin and their roles in the cell surface localization and transport function of P-glycoprotein in human colon adenocarcinoma LS180 cells." PloS one 16.5 (2021): e0250889.

• Shimomura, Seiji, et al. "Mechanical stimulation of chondrocytes regulates HIF-1α under hypoxic conditions." Tissue and Cell (2021): 101574.

• Morimoto, Hiromu, et al. "Progesterone depletion results in Lamin B1 loss and induction of cell death in mouse trophoblast giant cells." Plos one 16.7 (2021): e0254674.

• Someda, Masataka, et al. "Caspase-8, receptor-interacting protein kinase 1 (RIPK1), and RIPK3 regulate retinoic acid-induced cell differentiation and necroptosis." Cell Death & Differentiation 27.5 (2020): 1539-1553.

• Shibuya, Shuichi, et al. "Acai Extract Increases the Red Blood Cell Population via Erythropoietin Upregulation in Mice." (2020).

• Nishimoto, Yutaka, et al. "Carboxyl-, sulfonyl-, and phosphate-terminal dendrimers as a nanoplatform with lymph node targeting." International Journal of Pharmaceutics (2020): 119021.

• Shibuya, Shuichi, et al. "Acai Extract Transiently Upregulates Erythropoietin by Inducing a Renal Hypoxic Condition in Mice." Nutrients 12.2 (2020): 533.

• Lee, Sungho, et al. "Bone apatite anisotropic structure control via designing fibrous scaffolds." RSC Advances 10.23 (2020): 13500-13506.

• Hosokawa, Kohei, et al. "The lysosome pathway degrades CD81 on the cell surface by poly-ubiquitination and clathrin-mediated endocytosis." Biological and Pharmaceutical Bulletin 43.3 (2020): 540-545.

• Hisaoka-Nakashima, Kazue, et al. "Corticosterone Induces HMGB1 Release in Primary Cultured Rat Cortical Astrocytes: Involvement of Pannexin-1 and P2X7 Receptor-Dependent Mechanisms." Cells 9.5 (2020): 1068.

• Ohta, Ken-ichi, et al. "The effects of early life stress on the excitatory/inhibitory balance of the medial prefrontal cortex." Behavioural brain research 379 (2020): 112306.

• Kawarasaki, Satoko, et al. "A new mouse model for noninvasive fluorescence‐based monitoring of mitochondrial UCP 1 expression." FEBS letters 593.11 (2019): 1201-1212.

• Hisaoka-Nakashima, Kazue, et al. "Corticosterone Induces HMGB1 Release in Primary Cultured Rat Cortical Astrocytes: Involvement of Pannexin-1 and P2X7 Receptor-Dependent Mechanisms." Cells 9.5 (2020): 1068.