ProLong™ Live Antifade Reagent: Minimize photobleaching in live cells

ProLong™ Live Antifade Reagent helps reduce the loss of fluorescence that occurs with photobleaching during live-cell imaging. When illuminated, fluorescent dyes can degrade, reducing fluorescence intensity and leading to the creation of singlet oxygen (1O2), a highly reactive excited state of oxygen that can further degrade neighboring dye molecules. ProLong™ Live Antifade Reagent metabolizes 1O2, helping to maintain the intensity and duration of sample fluorescence. Moreover, we have observed little to no effect of ProLong™ Live reagent on cell viability and proliferation in experiments lasting up to 48 hours. Benefits of ProLong™ Live Antifade Reagent include:

  • Reduced photobleaching in live-cell experiments with fluorophores and fluorescent proteins across the spectrum
  • Extended imaging times in time-lapse experiments
  • Ease of use—just add to the imaging solution, complete cell culture medium, or buffer, and then incubate and image
Time lapse images of live cells showing photobleaching protection by ProLong Live Antifade Reagent  
Photobleaching protection by ProLong™ Live Antifade Reagent in live cells. HeLa cells were transduced with CellLight™ Mitochondria-GFP for 24 hr, then labeled with Hoechst™ 33342 for 15 min. Samples incubated with ProLong™ Live Antifade Reagent for 2 hr retained more signal at all time points (A), when compared with control samples in complete medium alone (B).

Foxp3 Transcription Factor Staining Buffer Kits

The Foxp3 Transcription Factor Staining Buffer Kits provide the fixation and permeabilization buffers needed for easy detection of intracellular Foxp3 transcription factor using fluorescent antibody conjugates. The buffer systems are available with either APC-, FITC-, or PE-conjugated anti–mouse Foxp3 monoclonal antibody (clone 3G3); staining buffers are also available separately.

Multiplexable Click-iT™ Plus TUNEL Assay for in situ detection of apoptosis

The Click-iT™ Plus TUNEL Assay detects apoptotic cells in tissues and cultured cells through the use of a small, highly specific labeling moiety and a brightly fluorescent dye. Once the alkyne-modified dUTP is incorporated into DNA fragments, detection is achieved with an Alexa Fluor™ azide through a catalyzed “click” reaction using conditions mild enough to preserve the fluorescent signal from GFP or RFP. The Click-iT™ Plus TUNEL Assay has been validated with several different formalin-fixed, paraffin-embedded tissue types; in all cases, the ability to multiplex with fluorescent proteins and dyes was preserved.

Multiplexing HeLa cells with the Click-iT Plus TUNEL Assay   Multiplexing with the Click-iT™ Plus TUNEL Assay. HeLa cells transduced with CellLight™ Mitochondria-RFP, BacMam 2.0 and treated with DNase to induce DNA strand breaks were analyzed using the Click-iT™ Plus TUNEL Assay (with Alexa Fluor™ 647 dye) to detect fragmented DNA (purple). ActinGreen™ 488 ReadyProbes™ Reagent was used to label actin filaments (green), and RFP expression was localized to mitochondria (red).

Superclonal™ secondary antibodies: Minimize cross-reactivity and background in secondary antibody detection

Thermo Scientific™ Superclonal™ secondary antibodies represent a breakthrough in recombinant antibody technology. Superclonal™ secondary antibodies are designed to provide precise and accurate detection of mouse, rabbit, and goat primary antibodies in ELISA, western blot, and cell imaging applications.

To produce Superclonal™ secondary antibodies, we employ a proprietary screening and production process that yields specific mixtures of recombinant goat or rabbit secondary antibodies. These antibodies bind with the epitope-specific precision of monoclonal antibodies, while also achieving the multi-epitope coverage (e.g., both heavy and light chains of target IgGs) and sensitivity of polyclonal antibodies. Superclonal™ secondary antibodies are available unconjugated, as well as conjugated with biotin, HRP, and select Alexa Fluor™ dyes.

HeLa cells showing reduction in nonspecific staining with Superclonal secondary antibodies   Reduction in nonspecific staining with Superclonal™ secondary antibodies. HeLa cell nucleoli were labeled with anti-nucleostemin primary antibody, which was then detected with the Alexa Fluor™ 488 conjugate (green) of (A) highly cross-adsorbed goat anti–mouse IgG (H+L) antibody, or (B) Superclonal™ goat anti–mouse IgG (H+L) antibody. Superclonal™ secondary antibodies exhibit significantly less cytoplasmic staining, indicating enhanced specificity.

Image-iT™ Hypoxia Reagent: A real-time oxygen sensor for live cells

Image-iT™ Hypoxia Reagent is a fluorogenic, cell-permeant compound for measuring hypoxia in live cells. This reagent is nonfluorescent in an environment with normal oxygen concentrations and becomes increasingly fluorescent as oxygen levels are decreased. Because it responds quickly to a changing environment, Image-iT™ Hypoxia Reagent can serve as a real-time oxygen detector, with a fluorescent signal that increases as atmospheric oxygen levels drop below 5% and decreases if oxygen concentrations increase. These properties make this reagent an ideal tool for detecting hypoxic conditions in tumor cells, 3D cultures, spheroids, neurons, and other live samples. Image-iT™ Hypoxia Reagent is very easy to use; just add it to cell culture medium and image.

4-panel image showing the fluorescence response of cells labeled with the Image-iT Hypoxia Reagent as oxygen levels drop   Fluorescence response of cells labeled with the Image-iT™ Hypoxia Reagent as oxygen levels drop. A549 cells were left overnight under normoxic conditions in a CO2 incubator and then treated with 5 μM Image-iT™ Hypoxia Reagent (red) and NucBlue™ Live Cell Stain (blue) in complete growth medium on an EVOS™ Onstage Incubator at varying O2 concentrations (20%, 5%, 2.5%, and 1.5%) for 1 hr and imaged on the EVOS™ FL Auto Imaging System.

WesternDot™ primary antibody conjugates for mCherry fusions and His-Tag labels

WesternDot™ primary antibodies are designed to reliably detect mCherry or His-Tag proteins in western blot applications, eliminating the need for secondary antibodies. These intensely fluorescent antibodies enable superior signal-to-noise ratios, as well as detection sensitivities that are comparable with those of ECL-based chemiluminescent methods. Different fluorescent colors of WesternDot™ antibodies can be applied to a single blot and multiple proteins detected simultaneously using standard gel or blot imaging platforms; no stripping and reprobing is required. It’s simple to try WesternDot™ antibodies in your current western blot applications; they are compatible with standard membranes, blocking solutions, and buffers.

gel image showing simultaneous detection of three different proteins on a single blot using WesternDot antibody conjugates   Simultaneous detection of three different proteins on a single blot using WesternDot™ antibody conjugates. A western blot containing mCherry and GFP lysate from HeLa cells spiked with decreasing quantities of His-tagged protein was probed with WesternDot™ 655 anti–His-Tag (red), WesternDot™ 800 anti-mCherry (blue, Cat. No. W10831), and chicken anti-GFP in conjunction with WesternDot™ 585 goat anti-chicken secondary antibody (green). The blot was imaged using the Fujifilm™ LAS-4000 gel imager.