Alexa Fluor® 488 dye–conjugated primary antibodies for tight junctions and the cytoskeleton 

what they are
Invitrogen offers one of the most extensive portfolios of antibodies for cell junctions and the cytoskeleton. This month, we are releasing new Alexa Fluor® 488 conjugates for key cell junction and cytoskeletal targets.

how they work

Alexa Fluor® 488 (excitation/emission 494/517 nm) is one of the brightest dyes available for imaging applications. Its conjugation to highly specific antibodies results in bright, crisp images with almost no background, and bypasses the need for secondary antibody detection. The new Alexa Fluor® conjugates of antibodies against occludin, claudin-1, claudin-4, claudin-5, and α–tubulin are valuable tools for imaging tight junctions and the cytoskeleton, allowing easy colocalization with other key markers.

what they offer
  • Save time and costs by eliminating secondary antibody detection
  • Ideal for imaging multiple targets in colocalization studies
  • Validated against multiple applications

Phases of Mitotic Cell
Human Caco-2 cells stained with mouse anti-α–tubulin Alexa Fluor® 488 (Cat. no. 322588), showing different phases of mitotic cell division. DNA is counterstained with blue Hoechst 33258 (Cat. no. H3569).


Product Quantity Applications* Species reactivity† Cat. no.
Mouse anti-claudin-1, Alexa Fluor® 488 conjugate100 µgELISA, ICC, IF, IP, WBCn, Hu, Ms, Rt374988
Mouse anti-claudin-4, Alexa Fluor® 488 conjugate100 µgICC, IFCn, Hu, Ms, Rt329488
Mouse anti-claudin-5, Alexa Fluor® 488 conjugate100 µgELISA, ICC, IF, IP, WBHu, Ms, Rt352588
Mouse anti-occludin, Alexa Fluor® 488 conjugate100 µgELISA, ICC, IF,  WBCn, Hu, Ms, Rt331588
Mouse anti-α-tubulin, Alexa Fluor® 488 conjugate100 µgIF, ICCHu, Ms, Rt322588
* ICC = immunocytochemistry, IF = immunofluorescence,
IP = immunoprecipitation, WB = western blot.
† Cn = canine, Hu = human, Ms = mouse, Rt = rat.

To browse antibodies by specificity or application, visit www.invitrogen.com/antibodies.
Click-iT® EdU assays: Detect plant DNA synthesis without cell wall digestion

what it is
Click-iT® EdU assays provide a fast, easy, and accurate method for detecting cell proliferation. Click-iT® assays use a modified nucleoside, EdU, which is incorporated during DNA synthesis. Unlike assays using BrdU, Click-iT® assays are not antibody-based and therefore do not require harsh DNA denaturation or cell wall digestion, which can compromise sample integrity and reliability.

how it works
Cell walls act as a barrier to detecting cell proliferation in plants. Using antibodies to detect plant DNA synthesis requires cell wall digestion, and the enzymes used often contain impurities that can decrease the reliability of the assay. Furthermore, the additional steps required can make EdU assays labor-intensive and time-consuming. Click-iT® EdU assays in plants involve only a mild fixation and permeabilization step, providing a significantly faster, more accurate method for detecting plant cell proliferation.

what it offers

  • Simple protocol—no denaturation or cell wall digestion
  • Reliable results—no exposure to enzyme impurities

Medicago sativa (alfalfa) Detection of DNA synthesis in undigested Medicago sativa (alfalfa) suspension cultures with Click-iT® EdU (green). Nuclei are counterstained with DAPI (pseudocolored black). Six confocal sections were merged into a differential interference contrast image.


Product Quantity Cat. no.
EdU (5-ethynyl-2´-deoxyuridine)50 mg A10044
EdU (5-ethynyl-2´-deoxyuridine)500 mg E10187
Click-iT® EdU Alexa Fluor® 488 Imaging Kit1 kit C10083
Click-iT® EdU Alexa Fluor® 594 Imaging Kit1 kit C10084
Click-iT® EdU Alexa Fluor® 647 Imaging Kit1 kit C10085

To learn more about EdU assays, visit www.invitrogen.com/edu.

Visualize global RNA transcription using robust click chemistry

what it is
5-ethynyl uridine (EU) is a novel uracil alternative to 5-bromouridine (BrU or BrUTP) for monitoring global RNA transcription temporally and spatially in vitro and in vivo.

how it works

Detection of incorporated EU is based on a simple and robust two-step technique that utilizes the chemoselective ligation or “click” reaction between an azide and an alkyne. In the first step, the EU containing an alkyne is administered to cells or animals. Following mild fixation and permeabilization, any incorporated EU is detected with a corresponding azide-containing detection reagent (including the bright and photostable Alexa Fluor® dyes) together with the Click-iT® Cell Reaction Buffer Kit (Cat. no. C10269).

what it offers

  • Compatible with multiple platforms—cells or tissue labeled with EU can be subsequently analyzed by fluorescence microscopy or high-content imaging
  • Simple and fast method—reaction is complete in 15–30 minutes, without long primary or secondary antibody incubations 
  • Sensitive—label and detection tags react selectively and specifically, providing extremely low background


U2OS cells
RNA synthesis in U2OS cells. Cells were treated with DMSO control (A) or 1 mM 5-ethynyl uridine (EU) (Cat. no. E10345) (B) for 1 hr. Cells were then fixed and permeabilized, and EU that had been incorporated into newly synthesized RNA was detected using green-fluorescent Alexa Fluor® 488 azide (Cat. no. A10266) and the Click-iT® Cell Reaction Buffer Kit (Cat no. C10269). Tubulin was labeled with mouse anti-α-tubulin antibody (Cat. no. A11126) and visualized with red-fluorescent Alexa Fluor® 594 goat anti-mouse IgG antibody (Cat. no. A11032). Nuclei were stained with blue-fluorescent Hoechst 33342 (Cat. no. H3570).

Product Quantity Cat. no.
5-ethynyl uridine (EU)5 mgE10345
Click-iT® Cell Reaction Buffer Kit1 kitC10269
Alexa Fluor® 488 Azide0.5 mgA10266


Learn More about click chemistry.

See bacterial communities in living color with FilmTracer™ biofilm stains

what they are
The new FilmTracer™ fluorescent stains promise to accelerate biofilm research by making it easier to examine bacteria within biofilm communities. Until now, biofilms have been difficult to stain because of their complex composition and structure. The FilmTracer™ stains have been tested and optimized specifically for imaging biofilms. 

how they work
A variety of FilmTracer™ stains are available for imaging different components of a biofilm. The FilmTracer™ FM® 1-43 dye stains the cells in a biofilm, whereas FilmTracer™ SYPRO® Ruby reagent stains the matrix. FilmTracer™ calcein reagents detect both cellular and matrix esterase activity within biofilms. The FilmTracer™ LIVE/DEAD® kit distinguishes live cells from dead cells within a biofilm. The stains are compatible with biofilm samples that are grown in the laboratory or collected from the environment (rocks, pipes, or catheters). Visualization by confocal laser scanning microscopy gives the best 3D readout of the stained biofilm.

what they offer

  • Easy protocol—just add stain, incubate, and observe
  • Flexible—monitor multiple aspects of biofilm biology
  • Validated—protocols tested specifically with biofilms

 

FilmTracer™ calcein red-orange FilmTracer™ calcein red-orange AM applied to a Staphylococcus epidermidis biofilm. The esterase substrate FilmTracer™ calcein red-orange AM appears to stain all of the bacteria in the biofilm, suggesting that the bacteria are actively producing esterase. The image was obtained using a Leica TCS-SP2 AOBS confocal microscope with a 63x/0.9 NA water immersion objective.

 

Product Quantity Cat. no.
FilmTracer™ LIVE/DEAD® Biofilm Viability Kit1 kitL10316
FilmTracer™ FM® 1-43 green biofilm cell stain1 mgF10317
FilmTracer™ SYPRO® Ruby biofilm matrix stain200 mLF10318
FilmTracer™ calcein red-orange biofilm stain20 x 50 µgF10319
FilmTracer™ calcein violet biofilm stain20 x 25 µgF10320
FilmTracer™ calcein green biofilm stain20 x 50 µgF10322

 

Live-cell imaging of focal adhesions

what they are
Cellular Lights™ reagents utilize fluorescent protein–signal peptide fusions for accurate and specific targeting to subcellular structures in live cells. Their high spatial and temporal resolution enable unique and powerful insights into biological systems without modifying cell function. Cellular Lights™ Talin-GFP and Talin-RFP, the newest additions to this powerful series of reagents, facilitate the visualization of focal adhesions in live cells via talin, a protein that links actin to the extracellular matrix.

how they work
Cellular Lights™ reagents are based on a baculovirus that, upon entry into a mammalian cell using BacMam technology, directs the expression of autofluorescent proteins to specific subcellular compartments by means of a well-documented targeting domain. Cellular Lights™ reagents are prepackaged as baculoviral particles for convenient transduction of a broad range of mammalian cell types, including primary and stem cells, without the need for lipids or dye-loading protocols that can perturb cell growth and viability.

what they offer

  • Ready-to-use reagents—no need to purify plasmids or prepare transfection complexes; just add to cells, wash, and visualize results the next day
  • Content-rich results— multiplex with other Organelle Lights™ and Cellular Lights™ fluorescent proteins, fluorescent dyes, or Qdot® nanocrystal conjugates
  • Context-rich results—study dynamic cellular processes in live cells with precise spatial and temporal resolution in addition to performing fixed-cell analyses


Cellular Lights™ Talin-GFP
Live-cell imaging with Cellular Lights™ Talin-GFP and Cellular Lights™ Talin-RFP. (A) HeLa cells were transduced with Cellular Lights™ Talin-GFP and Actin-RFP. Imaging was performed on live cells using a DeltaVision® Core microscope and standard FITC/TRITC filter sets. (B) HeLa cells were transduced with Cellular Lights™ Talin-RFP and Organelle Lights™ Mito-GFP and co-stained with 1 µg/mL Hoechst 33342. Imaging was performed on live cells using a DeltaVision® Core microscope and standard DAPI/FITC/TRITC filter sets.

Product Quantity Cat. no.
Cellular Lights™ Talin-GFP1 kitC10323
Cellular Lights™ Talin-RFP1 kitC10324


Learn more about Cellular Lights™ reagents.

Streptavidin conjugates

Secondary detection—many researchers immediately think of secondary antibodies. But don’t forget that streptavidin-biotin systems can be used for detection in much the same way as secondary antibodies for western blots, imaging, flow cytometry, microarrays, and other applications. Invitrogen offers a wide range of streptavidin conjugates, including our exceptional Alexa Fluor® dyes as well as classic conjugates such as R-phycoerythrin (R-PE) and horseradish peroxidase (HRP). Our conjugation processes are tightly controlled to provide a high degree of batch-to-batch consistency, and each product is backed by our experienced technical support staff. We also offer conjugates of avidin and NeutrAvidin—a modified avidin processed to remove its carbohydrates and lower its isoelectric point—for reduced background staining.

Bovine pulmonary artery endothelial cells Microtubules of bovine pulmonary artery endothelial cells. Cells were labeled with mouse monoclonal anti–α-tubulin antibody (Cat. no. A11126), followed by biotin-XX goat anti-mouse IgG antibody (Cat. no. B2763), and then visualized with Marina Blue® streptavidin (Cat. no. S11221). The cells were next treated with RNase, and the chromosomes were labeled with TO-PRO®-3 iodide (Cat. no. T3605). A series of Z-plane images was acquired with a wide-field optical sectioning confocal laser-scanning microscope. A three-dimensional volume rendering was generated from the deconvolved image series.

Learn more about our streptavidin, avidin, and NeutrAvidin products



Amplex® UltraRed reagent—a change for the brighter

Amplex® UltraRed reagent is our best soluble fluorogenic substrate for peroxidase activity. In the presence of horseradish peroxidase (HRP), Amplex® UltraRed reagent reacts with hydrogen peroxide to produce highly fluorescent UltroxRed. Amplex® UltraRed reagent improves upon the performance of Amplex® Red reagent, offering brighter fluorescence and enhanced sensitivity in peroxidase or peroxidase-coupled enzyme assays.

Amplex® UltraRed reagent is less sensitive to pH than Amplex® Red reagent, and exhibits improved stability in the presence of hydrogen peroxide (H2O2) or thiols such as dithiothreitol (DTT). Its stability makes Amplex® UltraRed the reagent of choice when working with cell lysates, where pH and redox potential are unknown or variable.

Change today for a brighter tomorrow—most of our well-cited Amplex® Red reagent–based, enzyme-coupled analyte assays come standard with fluorogenic Amplex® Red reagent as the substrate. However, switching any of the 14 existing Amplex® Red Assay Kits with Amplex® UltraRed reagent can result in signal enhancement of up to a 5-fold increase. 
 
Amplex® Ultra Red Amplex® UltraRed reagent is brighter than Amplex® Red reagent when using whole cell lysates. Detection of HRP using Amplex® UltraRed reagent (red) and the original Amplex® Red reagent (blue). Reactions contain 50 µM Amplex® UltraRed reagent or Amplex® Red reagent, and the indicated concentration of HRP in 100 mM HEPES, pH 7 with Jurkat cell lysates for 30 min at room temperature. Fluorescence was measured with a fluorescence-based microplate reader using excitation at 530 ± 12.5 nm and detection at 580 ± 25 nm. Background fluorescence was determined for a no-HRP control.


Product Quantity Cat. no.
Amplex® UltraRed reagent5 x 1 mg vialsA36006
Amplex® Red/UltraRed stop reagent500 testsA33855

Learn more about Amplex® UltraRed technology.

Simultaneous prospective purification of adult subventricular zone neural stem cells and their progeny.
Pastrana, E. et al. (2009) Proc Natl Acad Sci U S A (epub ahead of print).

What biochemical characteristics distinguish neural stem cells from their nonproliferative neighbors? Despite their relative rarity, neural stem cells serve the vital function of generating new neurons to provide building blocks for neural circuitry. In the adult mammalian brain, neural stem cells represent a subset of astrocytes that exhibit few cellular markers to distinguish them from nonproliferative astrocytes, making their isolation and study difficult. However, a subpopulation of stem cell astrocytes has been shown to express the epidermal growth factor receptor (EGFR); in the present study, Pastrana and colleagues utilized Alexa Fluor® dye–labeled EGF, combined with FACS analysis, to identify this subpopulation, and demonstrated that it represents activated stem cell astrocytes through subsequent treatment with an antimitotic drug. The researchers were also able to combine this analysis with Vybrant® DyeCycle™ dye staining, which allowed the sorting of cells according to their stage in the cell cycle. Further immunostaining and flow cytometric analysis revealed that the isolated subpopulation was as much as 20-fold enriched in cells exhibiting self-renewal and multipotency potential. The authors suggest that this simple purification strategy will greatly facilitate future molecular and functional studies of stem cell astrocytes, in pursuit of a greater understanding of stem cell behavior in vivo.

View bibliography reference.

 

Human osteosarcoma cell Human osteosarcoma cell (U2OS) visualized using fluorescent protein–based
Cellular Lights™ reagents.
Cells were transduced with Cellular Lights™ Histone
2B-RFP
(Cat. no. C10129) and Cellular Lights™ MAP4-GFP (Cat. no. C10105).
Cells were imaged the following day using a DeltaVision® Core microscope with
FITC/TRITC filter sets. The image shows a cell undergoing mitosis; condensed
histones (red) are clearly visible and are aligned parallel with the microtubules (green) that form
the mitotic spindle. Image contributed by Nicholas Dolman, Life Technologies Corporation. 


Fluorescence microscopy New web resource for fluorescence microscopy and immunofluorescence
We are working hard to update our website with improved content and utility for our customers. Recently, a number of pages related to fluorescence microscopy and immunofluorescence have been revamped. Now it’s easier than ever to find key products and information for fluorescence imaging, including quick links to secondary detection reagents, organelle stains, antifade reagents, and tools for automated imaging. 

You can also find information about some of the most advanced imaging systems available from Leica Microsystems to complement Invitrogen’s extensive array of imaging reagents.

Visit our new pages at www.invitrogen.com/immunofl and look for more improvements and updates soon.

High Content Screening - HCS Free online technical webinars
You are invited to join us for a series of biweekly technical webinars from the comfort of your desk. The webinars will initially focus on imaging-related applications, but we welcome your feedback for additional topics throughout the course of the year. Upcoming topics will be announced each month via email.

Presentations will last approximately 45 minutes, followed by 15 minutes for live Q&A.
Webinars Date Time
Organelle Stains and Labels
April 28, 2009 10:00 a.m. PT
Quantum Dots Insights
May 12, 2009 10:00 a.m. PT


Missed our previous webinars? Find them at the links below:



Molecular Probes® The Handbook

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