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Multiplexing with primary antibodies from the same species using Tyramide SuperBoost Signal Amplification

The ability to perform IHC using two primary antibodies from the same species opens up new possibilities for multiplex labeling. Combining the brightness of Invitrogen Alexa Fluor dyes and trusted poly-horseradish peroxidase mediated tyramide signal amplification, the recently developed Invitrogen Tyramide SuperBoost Signal Amplification kits not only provide enhanced sensitivity of low-abundance targets 10 to 200 times that of standard IHC methods, but also allow multiplex detection of primary antibodies from the same species on a tissue sample without risking antibody cross-reactivity.

Tyramide signal amplification is an enzyme-mediated detection method that utilizes the catalytic activity of horseradish peroxidase to generate high-density labeling of a target protein or nucleic acid sequence in situ. Tyramide SuperBoost technology produces highly reactive tyramide radicals that covalently react to localize antibody staining in IHC samples. Primary and secondary antibodies can thus be removed from the tissue using a citrate buffer/microwave method after the tyramide reaction without significantly decreasing the fluorescence intensity of the tyramide deposit. Once antibodies are removed, the tissue can be reprobed with a primary antibody from the same species without risk of cross-reactivity, followed by detection with another round of Tyramide SuperBoost signal amplification.

Sequential labeling and detection of three different rabbit primary antibodies using the Tyramide SuperBoost kits.

Sequential labeling and detection of three different rabbit primary antibodies using the Tyramide SuperBoost kits. A formalin-fixed, paraffin-embedded rat intestinal tissue section was sequentially labeled with rabbit anti-H2B antibody detected with the Alexa Fluor 647 Tyramide SuperBoost Kit (green), rabbit anti–smooth muscle actin antibody detected with the Alexa Fluor 488 Tyramide SuperBoost Kit (red), and rabbit anti-Ki-67 antibody detected with the Alexa Fluor 594 Tyramide SuperBoost Kit (blue). In between each antibody labeling, tissue samples were microwaved in citrate buffer, pH 6, on high power until boiling (~2 min), then microwaved for 15 min at 20% power, and finally allowed to cool to room temperature before subsequent labeling with the next rabbit antibody.

Learn more about Tyramide SuperBoost Signal Amplification and select kits for your multiplex labeling experiment at thermofisher.com/superboost


Recreating human skin—revolutionize air-liquid interface culture with the Nunc Carrier Plate and Insert system

Artificially grown skin models have become an important substitute for actual skin to simulate the effects of different conditions on epidermal tissue. Inducing keratinocytes to differentiate into the various layers of the epidermis requires direct exposure to air as well as to the culture media that supply the nutrients for cell growth and differentiation. The Thermo Scientific Nunc Cell Culture Inserts provide a porous membrane-based culture system with such air-liquid interface (ALI). The resultant skin models closely imitate the natural skin explants as demonstrated by the expression of markers of various differentiation stages.

The comparable expression of progenitor (K14), early (K10), intermediate (Involucrin), and late (Filaggrin) markers in differentiated 3D human epidermal models vs. natural skin explants.

The comparable expression of progenitor (K14), early (K10), intermediate (Involucrin), and late (Filaggrin) markers in differentiated 3D human epidermal models vs. natural skin explants. Dotted lines indicate insert membrane where the Human Epidermal Keratinocytes were seeded.


Combining the corresponding Nunc Carrier Plate with its multiple hanging positions for the inserts, medium feeding intervals can be tailored to better suit lab schedule and workload, offering flexibility in the experimental process.

Depiction of cell culture inserts hanging in three different positions in a carrier plate

Depiction of cell culture inserts hanging in three different positions in a carrier plate.


Make your own antibody dye conjugates using the SiteClick antibody labeling system

This modular, click chemistry–mediated method allows you to enzymatically label essentially any antibody on its heavy chain N-linked glycans. In contrast to standard antibody labeling techniques, which can be tedious and inconsistent, the SiteClick site-selective approach produces highly robust and reproducible labeling of antibodies with an impressive choice of detection molecules.

This new system allows simple and gentle site-selective attachment of detection molecules to heavy chain N-linked glycans—far from the antigen-binding domain—providing excellent reproducibility from labeling to labeling and from antibody to antibody. A number of different detection molecules can be site-selectively attached to the heavy chain glycans—including phycobiliproteins (e.g., R-PE), Qdot probes, Alexa Fluor dyes, metal-chelating compounds, and other small molecules like biotin—allowing multiplex analysis with antibodies from the same species.

  • Quality conjugates—enzymatic labeling system keeps antibodies and antigen-recognition site intact
  • Simple—Easy-to-follow step-by-step protocol—no chemistry skills needed
  • Reproducible—antibodies are labeled the same every time
 Immunochemistry with SiteClick labeled antibodies

Immunochemistry with SiteClick labeled antibodies. HeLa cells were fixed, permeabilized, and incubated with 10 nM Qdot 655 anti–golgin 97 antibody (magenta). This conjugate was generated using the SiteClick Qdot 655 Antibody Labeling Kit and mouse monoclonal anti–golgin-97 antibody (clone CDF3). After antibody incubation, the cells were counterstained with NucBlue Live (blue) and ActinGreen 488 (green) ReadyProbes reagents prior to imaging.


ProLong Glass antifade mountant: Get images at 3-4 more focal depth with uncompromised resolution

Tissue specimens are three dimensional, but light scattering and other phenomena makes tissues opaque for examination, and with conventional methods we can only examine samples that are a few µm thick. This forced us to cut the tissues into 5-8 µm thick sections, and much tissue biology was lost in between these sections, not to mention the amount of work required to process dozens of tissue sections if examination of 3D structure was required. It is now possible to examine tissues up to 100 µm thick, by using the newly developed Prolong Glass antifade mountant, with a refractive index (RI) of 1.52 (after curing). This RI is same as cover glass and immersion oil, minimizing light scattering, and letting fluorescent light penetrate deeper into the tissue.

Mismatch of RI between mounting media, cover glass and immersion oil, causes spherical aberrations, making it difficult for microscope objectives to focus on a specific object, resulting in reduced axial resolution.  This scattering of light also reduces the amount of fluorescence light that can make it back to objective, reducing intensity with increasing focal depth.

With increased laser intensity, objects up to 150 µm thick can also be been imaged with high resolution. When using Prolong Glass antifade mountant with an RI of 1.52, the pig brain nuclei are roundish in shape, with sharp nuclear details. The figure below shows the increase in signal intensity achieved with increased focal depth, making it more likely to capture tissue/cellular biology at up to 100 µm thickness.

Axial (XZ) confocal image of FFPE pig brain sections, mounted with different commercial antifade formulations.

Axial (XZ) confocal image of FFPE pig brain sections, mounted with different commercial antifade formulations.

Select the Invitrogen ProLong or SlowFade antifade mountant that best matches your experimental needs.

Feature ProLong Glass ProLong Diamond ProLong Gold SlowFade Diamond SlowFade Gold
Hard/soft setting Hard-setting (curing) Soft-setting (non-curing)
Refractive index ~1.52 after curing (same as crown glass) ~1.47 after curing ~1.47 after curing ~1.42 ~1.42
Z-stacking, 3D reconstruction/deconvolution Yes Not recommended
Recommended objective type

Oil-immersion for best results. 

Other objective types are compatible.

Glycerol corrective objectives for best results. 

Other objective types are compatible.

Cell/tissue thickness 0–150 μm Best results from 0–10 μm. Imaging up to 30 μm is possible under optimized conditions.
Organic dye photobleach protection† +++ +++ ++ +++ ++
Fluorescent protein photobleach protection† +++ +++ Not recommended +++ Not recommended
Slide storage after mounting Long term (weeks to months) Short term (days to weeks)
Example skus P36980 P36961 P36934 S36963 S36937

Find out more at thermofisher.com/antifades

NEW PRODUCT RELEASES

New Invitrogen antibodies for IHC

Over 2,500 new Invitrogen antibodies were launched at the beginning of 2018, many of which had IHC data images. This brings the number of antibodies qualified for use in IHC to almost 40,000. With over 125,000 data images on the web demonstrating antibody performance, you can be confident when purchasing Invitrogen antibodies for your experiment. Supported by our Antibody Performance Guarantee, our antibodies will work as demonstrated on our website.

HDAC9 Antibody (MA5-26729) in IHC (P)

HDAC9 antibody (MA5-26729) in IHC (P). Immunohistochemistry was performed on paraffin-embedded human endometrium tissue. To expose target proteins, 1 mM EDTA in 10mM Tris, pH8.5, 120°C for 3min was used. Following antigen retrieval, tissues were probed with a HDAC9 monoclonal antibody (MA5-26729).

Find the antibody you need for IHC thermofisher.com/antibodies


New Invitrogen antibodies for flow cytometry

As a flow cytometry supplier, we are dedicated to helping you get to your science faster. In the past year we released >860 flow cytometry antibody conjugates to provide you with more options when designing your flow cytometry panels. Antibodies released in 2017 and so far in 2018, include:

  • >700 eBioscience Super Bright antibody conjugates for the violet laser
  • >160 antibody conjugates
  • 16 new clones including—
    • Anti-Human CD370 (Clec9A) (clone 9A11)
    • Anti-Human CD318 (CDCP1) (clone CSTEM26)
    • Anti-Human CD321 (F11R) (clone CSTEM27)
    • Anti-Human CD141 (thrombomodulin) (clone JAA17)
    • Anti-Mouse CD163 (clone TNKUPJ)
CD321-f11r-antibody

CD321 (F11R) antibody (12-3219-42). Staining of a mixture of human iPSC and the C2C12 murine cell line with Anti-Human TRA-1-60 PerCP-eFluor 710 (46-8863-82) and Mouse IgG2a K Isotype Control PE (12-4724-81) (left) or Anti-Human CD321 (F11R) PE (right).

Visit our updated Flow Cytometry web page and search the 10,000+ flow cytometry antibodies quickly and easily using our new catalog search tool at thermofisher.com/flowantibodies


Need a labeled antibody in a hurry? New Zip Antibody Labeling kits are the answer.

Zip Antibody Labeling kits

Not all antibodies are available as fluorescent conjugates for your flow or imaging experiment. Our new Zip kits allow you to efficiently label your precious antibody with Alexa Fluor dyes to create an antibody conjugate that is ready for use within 15 minutes.

  • Very fast and easy to use
  • 100% antibody recovery– no purification steps
  • Useful for a wide range of applications
  • No chemistry or conjugation experience needed

RESOURCES AND GUIDES

On-demand webinar: Five steps for publication-quality immunohistochemistry imaging

On-demand webinar: Five steps for publication-quality immunohistochemistry imaging

If you missed the live broadcast of this webinar on May 2nd, you can view it on-demand in two ways:

  • Directly via the Labroots microsite or
  • Via Protein and Cell Analysis Education Resources portal, where a larger list of Thermo Fisher Scientific on-demand webinars can be viewed: thermofisher.com/proteincelledu

Immunohistochemistry: Five steps to publication-quality images

Watch these two videos for tips to address weak/ no stain and high background problems for all five steps of the IHC workflow.

To find IHC antibodies click here


BioProbes Journal of Cell Biology Applications—Click-iT EdU and Click-iT TUNEL colorimetric assays for immunohistochemistry

Labeling of cells and tissues with several distinct fluorescent probes is now a standard lab practice that allows for multiplexing of functional and antibody-based markers. However, there are fields of research and types of analyses that are much more amenable to colorimetric labeling. Colorimetric detection is far more widespread when studying highly autofluorescent tissue, such as the heart, because autofluorescence can significantly interfere with a fluorescent probe signal. Colorimetric detection is also favored when labeling must be compatible with standard histological stains that provide morphological and contextual information. Here we describe the conversion of our popular fluorescence-based click chemistry assays for cell proliferation and apoptosis to colorimetric assays.

Mouse tissue section labeling with the colorimetric Click-iT TUNEL apoptosis assay

Mouse tissue section labeling with the colorimetric Click-iT TUNEL apoptosis assay. An 8 μm formalin-fixed, paraffin-embedded (FFPE) section of mouse thymus was labeled with DAB using the Click-iT TUNEL Colorimetric IHC Detection Kit to reveal apoptotic cells (dark brown nuclei). The tissue was subsequently stained with eosin Y (pink) followed by nuclear counterstaining with methyl green (blue), then dehydrated, and hard-mounted in Thermo Scientific Cytoseal 60 Mounting Medium. The brightfield image was acquired using a 20x objective on the EVOS FL Auto Imaging System equipped with a color camera.


High-content screening-based phenotypic analysis of organotypic 3D bronchial tissues

High Content Screening-Based Phenotypic Analysis Of Organotypic 3D Bronchial Tissues

We are delighted to notify you about our on-demand webinar in close collaboration with and presented by one of our CellInsight CX7 customers, Diego Marescotti from Phillip Morris International in Switzerland. This is a great opportunity for customers to learn from Diego and his team’s work about High-Content technology and 3D screening for toxicology assays and how it could benefit them as well.

Webinar abstract:

One of the goals of the “3Rs principle” is to replace in vivo testing methodologies with in vitro procedures on human cells, followed by computational systems biology modelling to determine toxicological risk. System toxicology, which integrates standard toxicology together with quantitative analysis of large networks of molecular and functional changes, have demonstrated the potential to help realize the 3Rs vision. Omics and high content screening methods represent fundamental pillars of a system toxicology-based approach and at Philip Morris International R&D we have developed a large battery of HCS assays to investigate how specific substances alter the phenotype of a cell grown in submerged conditions. As more physiological cell culture models, such as organotypic 3D culture are also part of our portfolio, we invested in the development of imaging-based approaches which would enable high-content investigations of 3D bronchial tissues. In this seminar, recently developed approach for phenotypic analysis of organotypic 3D bronchial tissues are presented.

Watch the on-demand webinar


Boost synaptic strength—complete the protein and cell analysis eLearning course series

Boost synaptic strength—complete the Protein and cell analysis eLearning course series

This series of courses was designed to provide engaging, interactive learning experiences relevant to protein and cell analysis application areas and topics. These free, self-paced, animated courses include knowledge checks and practical application exercises that enable you to demonstrate what you have learned by completing educational modules.

Series of courses:

  • T Cell Stimulation and Proliferation
  • Protein Sample Preparation
  • Antibody Validation* (newly released)
  • Cancer Spheroid and Organoid Models (coming soon)

All content is available 24 hours a day, 7 days a week, and is viewable from the convenience of your desk, tablet, or mobile device. See what it’s all about at thermofisher.com/elearningcourses

*The use or any variation of the word “validation” refers only to research use antibodies that were subject to functional testing to confirm that the antibody can be used with the research techniques indicated. It does not ensure that the product, or products were validated for clinical or diagnostic uses.

For research use purposes only


Discover the Protein Biology Learning Center

Protein Biology Learning Center

Learn about protein analysis methods and technologies

The emphasis of Protein biology is the study of the structure and function of proteins as the primary focus of investigation, and the use of antibodies, proteins, and peptides as tools to purify, detect, and characterize biological systems. This collection of methods articles, videos, white paper, and other resources provides detailed information about all of the major areas of protein analysis that include: protein extraction; protein isolation and purification; western blotting, ELISA, protein assays; protein mass spectrometry; and protein modification and crosslinking, and other application areas. To explore the Protein Biology Learning Center, visit, thermofisher.com/pblc

This learning center provides both new and experienced scientists with our many resources for learning about protein analysis methods and products. The representative example below shows striking immunohistochemistry (IHC) staining results obtained with Thermo Scientific Invitrogen antibodies and other IHC reagents. To view the entire collection of methods articles relevant to IHC, begin with the Overview of Immunohistochemistry (IHC).

Detection of HDAC4 in human skin by IHC

Detection of HDAC4 in human skin by IHC. Chromogenic IHC was performed on thin sections of human skin obtained from biopsies.  The sections were stained either with a rabbit polyclonal antibody against HDAC4 (PA1-863) or without this antibody (the negative control).  HDAC4 detection was performed using a biotinylated anti-rabbit IgG secondary antibody and streptavidin-Horseradish peroxidase (HRP), followed by colorimetric detection using DAB. The sections were then counterstained with hematoxylin and mounted under coverslips.  In the left hand panel above, the HDAC4 antigen is stained brown by the precipitated DAB reaction product.  The control section on the right is not stained brown because no anti-HDAC4 primary antibody was used. Only the blue hematoxylin counterstaining can be seen.

NEWS AND VIEWS

Improved experience on antibody web pages—published antibody data added to Product Data pages

In the last couple of years, the Invitrogen antibody customers have witnessed many improvements when navigating the web pages and searching for primary and secondary antibodies.

Just recently added, over 25,000 images across almost 4,000 primary antibodies now display an additional gallery on the product page, showing application figures from peer-reviewed journals. Some antibodies have as many as 40 images, across a spectrum of applications in which our antibodies are used.

Application images have the most impact when deciding what antibody to choose for a project or specific application. This type of information provides additional validation on the Invitrogen antibody performance.

On the product page, the published figures are featured in the “Published Antibody Data” gallery section, above the “Antibody Testing Data” section of the page.

Product Data pages

Each of the featured images, if clicked, lead to the BenchiSci web page, where zoom in or view the figure legend are available.

Additionally, the references listed at the bottom of the product page for Invitrogen antibodies, will also feature a thumbnail of the relevant figure and a hyperlink to view the published figure on BenchSci. When clicking on the article title, the hyperlink will lead to the relevant article on PubMed.

Look for more published figures covering more antibodies to be added over time.

Find the antibody you need and check if it has published figures at thermofisher.com/antibodies


Pathways, panel design and protocols for flow cytometry!

We have recently updated the Antibodies for Flow Cytometry webpage! Our new antibody search tool is front and center on the page, making it easier and faster to search and find the flow cytometry antibody that you need from our extensive portfolio of 10,000+ immunology antibodies. The new design provides helpful links to key biological pathways, our panel design tool, and also to key cell preparation and cell staining protocols.

Check it out at thermofisher.com/flowantibodies

Antibodies for Flow Cytometry webpage

Not for resale. Super Bright Polymer Dyes are sold under license from Becton, Dickinson and Company.