Conferences & Tradeshows
National Society for Histotechnology (NSH)
Birmingham, USA
October 4–6
Society for Neuroscience (SFN)
Chicago, USA
October 17–21
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- Read the latest issue of the ProbesOnline E-Newsletter.
In This Issue
FEATURED NEW PRODUCTS
Accurate Compensation Settings Every Time—AbC™ anti-Rat/Hamster Bead Kit
| what it is
| how it works
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Ultrasensitive cAMP Detection—cAMP Chemiluminescent Immunoassay Kit
| what it is
| how it works
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Picture-Perfect Imagery with Qdot® Nanocrystals—Qmount™ Qdot® Mounting Media
| what it is
| how it works
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Simultaneously Distinguish Early and Late Apoptotic Cells—Pacific Blue™ Annexin V/SYTOX® AADvanced™ Apoptosis Kit
| what it is
| how it works
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NEW APPLICATIONS
Fluorescent Alternatives to Chromogenic-Based Immunohistochemistry
Immunohistochemistry (IHC) protocols for tissues and tissue microarrays are dominated by approaches using enzyme-coupled secondary antibodies that deposit chromogenic substrates. Their long history, well-established workflows, and archival properties support this approach. Yet standard immunohistochemistry techniques are limited to semiquantitative analysis (i.e., 0, +1, +2, +3) and narrow dynamic range, as enzymatic reactions tend to saturate, and are most often limited to one or two analytes. This is a serious complication in cancer diagnostics as the number of biomarkers increases, demanding protocols that can analyze more and more analytes with improved quantitation.
Several recent publications [1–4] show that protocols that incorporate fluorescent organic dyes, inorganic Qdot® nanocrystals, superior antifade mountants, and intensity calibration standards can replace traditional chromogenic methods. Fluorescence-based methods provide many of the advantages of traditional approaches, including ease of use and stable signal amplification, but benefit from broader dynamic ranges and the improved resolving power of direct vs. enzyme conjugates. Fluorescent labels also demonstrate an extended storage life and offer the possibility of adding 2–5 or more separate labels.
Explore Invitrogen's antibody offerings for IHC and other applications.
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Fluorescent detection of E-cadherin, Ksp-cadherin, and collagen IV in kidney. 3 μm FFPE sections of adult human kidney were dewaxed, rehydrated, and subject to antigen retrieval. Sections were stained with anti–E-cadherin mAb and anti-Ksp–cadherin mAb or with anti-E-cadherin mAb and anti-collagen IV Ab followed by Alexa Fluor® 488–conjugated goat anti-mouse IgG2A and Alexa Fluor® 555 goat anti-mouse IgG1 or Alexa Fluor®488-conjugated goat anti-mouse IgG2A and Alexa Fluor ® 555 goat anti-rabbit Ig. Nuclei were counterstained with DAPI. Arrows indicate tubules expressing both cadherins; arrowheads indicate tubules expressing neither cadherin. * Indicates tubules expressing E-cadherin alone. Scale bar, 50 μm. Reproduced from BioMed Central: Robertson D, Savage K, Reis-Filho JS, Isacke CM (2008) BMC Cell Biol 19:9. |
References
1. BMC Cell Biol 19:9 (2008)
2. Cancer Epidemiol Biomarkers Prev 16:1371 (2007)
3. Nano Lett 6:2881 (2006)
4. Nat Protoc 2:1152 (2007)
| Product | Quantity | Cat. No. | |
|---|---|---|---|
| ProLong® Gold antifade reagent, special packaging | 5 x 2 mL | P36934 |  |
| ProLong® Gold antifade reagent | 10 mL | P36930 | |
| ProLong® Gold antifade reagent with DAPI, special packaging | 5 x 2 mL | P36935 | |
| ProLong® Gold antifade reagent with DAPI | 10 mL | P36931 | |
| Qmount™ Qdot® Mounting Media | 3 x 2 mL | Q10336 | |
| InSpeck™ Green (505/515) Microscope Image Intensity Calibration Kit, 2.5 µm | 1 kit | I7219 | |
| InSpeck™ Blue (350/440) Microscope Image Intensity Calibration Kit, 2.5 µm | 1 kit | I7221 | |
| InSpeck™ Orange (540/560) Microscope Image Intensity Calibration Kit, 2.5 µm | 1 kit | I7223 | |
| InSpeck™ Red (580/605) Microscope Image Intensity Calibration Kit, 2.5 µm | 1 kit | I7224 | |
| InSpeck™ Deep Red (633/660) Microscope Image Intensity Calibration Kit, 2.5 µm | 1 kit | I7225 | |
| InSpeck™ Green (505/515) Microscope Image Intensity Calibration Kit, 6 µm | 1 kit | I14785 | |
| InSpeck™ Orange (540/560) Microscope Image Intensity Calibration Kit, 6 µm | 1 kit | I14786 | |
| InSpeck™ Red (580/605) Microscope Image Intensity Calibration Kit, 6 µm | 1 kit | I14787 | |
DEPARTMENTS
Buzzworthy
EdU-based labeling reveals differences in how cell lines react to thymidine substitution
Cell type specific applicability of 5-ethynyl-2’-deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry.
Diermeier-Daucher S, Clarke ST, Hill D et al. (2009) Cytometry 75A:535–546.
Do all cell lines react the same to nucleoside analog–based labeling?
The nucleoside analog EdU (5-ethynyl-2’-deoxyuridine) offers significant advantages over BrdU for assessing the proliferation state and cell cycle status of cells, chief among them being the ability to detect the probe without denaturing the cellular DNA. However, the impact of EdU on cell viability and function has not been extensively characterized. In their current study, Diermeier-Daucher and colleagues examined the effect of pulse- and continuous labeling of two breast cancer cell lines (BT474 and SK-BR-3) with EdU as compared to the effect seen with BrdU labeling under the same conditions.
Differences in long- and short-term EdU exposure
Each nucleoside analog yielded essentially identical staining intensity. However, incubation with EdU revealed a dose- and time-dependent increase in necrotic cell death in SK-BR-3 cells following 96 hr exposure, and both cell lines showed an increase in cell cycle arrest following long-term (144 hr) exposure to EdU. No negative impact was observed with these cells following short-pulse labeling with EdU, illustrating its utility as a sensitive and quantitative probe for flow cytometric assessment of proliferation in these cells.
Conclusion
As previous studies have reported similar negative cellular effects associated with exposure to BrdU, the authors demonstrate the necessity of evaluating the individual response of a given cell line to nucleoside analog exposure prior to carrying out continuous labeling experiments.
View bibliography reference
- Learn More about Click-iT® EdU Cell Proliferation Assays
- See all products for Flow Cytometry
Online Technical Webinars
 | 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.
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Missed our previous webinars? Find our recorded webinars here!
 | Combining Qdot® nanocrystals, organic dyes, and antibodies for superior cellular imaging. Human carcinoma (HeLa) cells were labeled with mouse anti-OxPhos Complex V inhibitor protein IgG, Qdot® 625–conjugated goat anti-mouse IgG (red), rabbit anti-giantin (Covance Bio), Qdot® 585–conjugated goat anti-rabbit IgG (yellow), and rat anti-tubulin (Serotec). Secondary labeling was performed with anti–DSB-X biotin goat anti-rat IgG, and a tertiary streptavidin–conjugated Qdot® 525 (green). The cells were then labeled with Qnuclear™ Deep Red stain (purple) and mounted in Qmount™ Qdot® mounting media. |
Tracking Stem Cells With Qdot® Nanocrystals
Tracking the migration of stem cells to target tissues and analysis of their subsequent differentiation are critical to the development of therapeutic regimes for stem cell therapy. Once differentiated, stem cells lose all stem cell–associated markers as they blend into the resident environment; they must be labeled prior to migration to be followed through differentiation. In addition to MRI, expression tags (e.g., fluorescent proteins), and organic dye–based reporters (e.g., CM-DiI, CFSE), Qdot® nanocrystals are particularly useful for stem cell labeling because they provide an extremely bright and photostable signal, even into the near infrared. Qdot® nanocrystals are detectable for at least 14 days postinjection [1], and have demonstrated up to at least 8 weeks of stability in another study [2].
Qdot® nanocrystals also offer:
- Easy delivery to cells
- Stability through many rounds of cell division (up to 6 or more depending on detection method and loading levels)
- No effects on cell proliferation [2], pluripotency/differentiation, or animal physiology
- No transfer to neighboring cells
- Availability in many colors
Several recent publications highlight the utility of Qdot® nanocrystals in this essential application area [1–4]. Reported delivery methods vary, with one study [2] preferring 24-hour incubations with Qdot® ITK™ carboxyl nanocrystals, but most preferring to use the cell-penetrating peptide conjugates included in the Qtracker® Cell Labeling kits [1,3,4]. Jablonski et al. [4] have shown most recently that incubation of Qdot® nanocrystals with pyrenebutyrate allows passive diffusion of poly-Arg conjugated nanocrystals into cells, resulting in a much more uniform, non-granule staining pattern.
An important cautionary note: All Qdot®-labeled material needs to be mounted in Qmount™ Qdot® mounting media or PBS. Avoid mounting in any organic dye–based mountant such as ProLong® Gold.
References
1. BMC Biotechnol 7:67 (2007)
2. Stem Cells 25:2128 (2007)
3. Histochem Cell Biol 130:329 (2008)
4. J Phys Chem B 113:405 (2009)
- Learn More about Qdot® Nanocrystals
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Qtracker® quantum dot label reveals distribution of injected human mesenchymal stem cells. Qtracker® 655 non-targeted quantum dot–labeled human mesenchymal stem cells were injected into a canine heart ventricle in an effort to create a biological pacemaker. Injected cells (red labeled cells in panels A and B) were detected 1 day later in fixed and sectioned material at (A) low magnification and (B) high magnification along with blue Hoechst 33342 staining. Additional experiments have shown that stem cells labeled in this manner are visible after 8 weeks and migrate in a pattern that mimics the resident in situ myocardial orientation [2]. Image courtesy of Amy Rosen, Institute for Molecular Cardiology, State University of New York at Stony Brook. |
| Product | Quantity | Cat. No. | |
|---|---|---|---|
| Qtracker® 625 Cell Labeling Kit | 1 kit | A10198 | |
| Qtracker® 625 non-targeted quantum dots | 200 µL | A10199 | |
| Qtracker® 655 non-targeted quantum dots, 2 µM solution | 200 µL | Q21021MP | |
| Qtracker® 565 non-targeted quantum dots, 2 µM solution | 200 µL | Q21031MP | |
| Qtracker® 705 Cell Labeling Kit | 1 kit | Q25061MP | |
| Qtracker® 800 Cell Labeling Kit | 1 kit | Q25071MP | |
| Qtracker® 705 non-targeted quantum dots, 2 µM solution | 200 µL | Q21061MP | |
| Qtracker® 800 non-targeted quantum dots, 2 µM solution | 200 µL | Q21071MP | |
| Qtracker® 605 Cell Labeling Kit | 1 kit | Q25001MP | |
| Qtracker® 585 Cell Labeling Kit | 1 kit | Q25011MP | |
| Qtracker® 655 Cell Labeling Kit | 1 kit | Q25021MP | |
| Qtracker® 565 Cell Labeling Kit | 1 kit | Q25031MP | |
| Qtracker® 525 Cell Labeling Kit | 1 kit | Q25041MP | |
| Qmount™ Qdot® Mounting Media | 3 x 2 mL | Q10336 | |
 | New Web Resource for Click-iT® EdU Cell Proliferation Assays Click-iT® EdU Cell Proliferation Assays offer faster, easier detection of newly synthesized DNA in a variety of fluorescent colors. Our new web page devoted to these assays allows you to select the flow cytometry, imaging, or high-content screening kit that is best for your research.
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 | New Web Page Dedicated to Alexa Fluor® Dyes * Life Science Award
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Molecular Probes® The Handbook
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