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Intracellular Immunofluorescence Labeling Kit (INF-1)

Intracellular Immunofluorescence Labeling Kit (INF-1)
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Price: $350.00
Availability: In Stock
Model: INF-1
Manufacturer: Fivephoton Biochemicals

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Intracellular Immunofluorescence Cell Staining (Labeling) Kit
 
Highlights
  • Immunofluorescence labeling with membrane permeabilization by using saponin.
  • Applicable for multiple-simultaneous immunofluorescent labeling as well as labeling of single epitopes.
  • Facilitates labeling of intracellular organelles (i.e. Golgi, mitochondria, endoplasmic reticulum, lysosomes and nuclei). 
  • Stock solutions can be thawed and frozen multiple times, or aliquoted and frozen for later use.
  • Ideal for laboratory teaching as well as research applications.
        Suitable for 40 microscope slides
 

Protocol

Kit Contents
  1. Instruction Manual
  2. 10X PBS pH 7.4;  16 ml
  3. 10X Quenching Buffer;  25 ml
  4. 10X Permeabilization Buffer;  8 ml
  5. 10X Intracellular Detection Buffer;  60 ml
  6.  Forceps set
Immunofluorescence Labelling of Cells
The Fivephoton Biochemicals intracellular immunofluorescence staining kit enables simultaneous labelling of multiple intracellular epitopes.  The immunofluorescence kit uses bark saponin for membrane permeabilization, a reagent that permeates the outer cellular membrane as well as organeller membranes, and allows antibody to reach all cellular regions.  Saponin permeation faciliates high resoluion imaging since membraneous structures remain intact.  To exclusively label intracellular epitopes, use primary antibodies that recognize intracellular domains.

Immunofluorescence staining with membrane permeabilization is employed to visualize intracellular structures and organelles such as nuclei, actin filaments, Golgi, and endoplasmic reticulum.  Intracellular cytosolic and organellar protein epitopes as well as intracellular domains of transmembrane proteins are labeled with this kit since antibodies have access to all intracellular regions.

(An important experimental distinction is provided in the Fivephoton Biochemicals Extracellular Immunofluorescence Kit (Part No. EIF-1) which enables exclusive extracellular labeling.  The application in the Extracellular Immunofluorescence Labeling Kit does not include a membrane permeabilization reagent and therefore preserves membrane integrity).


Immunofluorescence Staining Protocol, Synopsis
Cells, grown on glass cover slips or slides, are first fixed in paraformaldehyde or methanol.  The reaction is quenched with the provided "Quenching Buffer."  Cells are then permeabilized with the provided "Permeabilization Buffer," and then incubated with antibodies using the included "Intracellular Detection Buffer." For multiple-labeling, up to three different antibodies generated in different hosts are added simultaneously in the primary antibody labeling step.  After washes of unbound antibody, cells are incubated in the dark with fluorophore conjugated secondary antibodies.  For multiple labeling, three matched pairs of secondary antibodies with different fluorescence tags are applied to the cells.  Subsequent washes remove unbound antibody.  Cover slips or slides are then mounted with an anti-fade solution.  The anti-fade solution is allowed to harden and the cover slips or slides are ready for fluorescent microscopy.

Multiple labeling immunofluorescence images developed with the Intracellular Immunofluorescence Kit (INF-1).  Red:  A cation channel.  Green:   An intracellular binding protein involved in membrane trafficking.  Blue:  DAPI staining of the nucleus. 

   
Figure legends for "Images and Data" Tab
Representative microscopy images derived by the FIVEphoton Intracellular Immunofluorescence Labeling Kit are displayed in the icon and "Images and Data" tab.  Microscopy images in the "Images and Data" tab are as follows:  1) An ABC transporter expressed by transfection in HEK cells: 2)  Example of multiple-labeling immunofluorescence of an amyloid protein and an endoplasmic reticulum marker expressed by transfection in COS-7 cells.
   

Cellular Regions and Membrane Topology

Representative References on Immunofluorescence Labeling
  1. Blot V and McGraw TE. Use of quantitative immunofluorescence microscopy to study intracellular trafficking: studies of the GLUT4 glucose transporter. Methods Mol Biol 457: 347-366, 2008.
  2. Constans J, Oksman F, and Viau M. Binding of the apo and holo forms of the serum vitamin D-binding protein to human lymphocyte cytoplasm and membrane by indirect immunofluorescence. Immunol Lett 3: 159-162, 1981.
  3. Crivellato E, Travan L, Candussio L, Bartoli Klugmann F, and Decorti G. Identification of P-glycoprotein at the membrane of mast cell secretory granules. An immunofluorescence and protein A-gold electron microscopical investigation. Histochem J 29: 193-198, 1997.
  4. Doody A and Putnam D. Identification of compartments involved in mammalian subcellular trafficking pathways by indirect immunofluorescence. Methods Mol Med 127: 127-136, 2006.
  5. Fleischmajer R, Dessau W, Timpl R, Krieg T, Luderschmidt C, and Wiestner M. Immunofluorescence analysis of collagen, fibronectin, and basement membrane protein in scleroderma skin. J Invest Dermatol 75: 270-274, 1980.
  6. Gajl-Peczalska K. Plasma Protein Composition Of Hyaline Membrane In The Newborn As Studies By Immunofluorescence. Arch Dis Child 39: 226-231, 1964.
  7. Lejneva OM, Abelev GI, Dorfman NA, Strand M, and August JT. Localization of a murine oncornavirus 15,000-dalton virion protein on the membrane of neoplastic cells: analysis by immunofluorescence and immunoelectron microscopy. Virology 75: 281-292, 1976.
  8. Risau W, Saumweber H, and Symmons P. Monoclonal antibodies against a nuclear membrane protein of Drosophila. Localization by indirect immunofluorescence and detection of antigen using a new protein blotting procedure. Exp Cell Res 133: 47-54, 1981.
  9. Tezuka T and Takahashi M. The 55-kd keratohyalin granule protein has the same epitope as the 43-kd stratum corneum membrane protein: immunofluorescence and immunoblotting studies using a monoclonal antibody to the 55-kd keratohyalin granule protein. Arch Dermatol Res 280: 462-468, 1989.
  10. Wang H, Lee EW, Cai X, Ni Z, Zhou L, and Mao Q. Membrane topology of the human breast cancer resistance protein (BCRP/ABCG2) determined by epitope insertion and immunofluorescence. Biochemistry 47: 13778-13787, 2008.
Safety: Contains Irritants. Avoid ingestion, eye and skin contact.
Storage: Shipped at ambient temperature.  Store at -20oC.  Solutions can be thawed and frozen multiple times.
Shipping Options:  Fedex 2-3 day domestic delivery or overnight delivery.  Contact salessupport@FIVEphoton.com for other delivery options, including international delivery.

 


 

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Protocol Manual click here
MSDS click here



 

Intracellular Immunofluorescence Labeling Kit (INF-1)
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Intracellular Immunofluorescence Labeling Kit (INF-1)
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