Antibody Insider—Issue 2018 Q1

Fluorescent Immunohistochemistry (IHC) is a simpler alternative to the conventional chromogenic IHC method, and has a few advantages: stained slides have a longer shelf life as the signal is more stable and also do not require complicated advanced equipment to visualize the result. The fluorescent IHC technique however has an edge over the traditional method of chromogenic IHC in the following ways: 

• It involves fewer steps; for example the addition of substrate as in the case of chromogenic methods is omitted
• Images with higher resolution can be obtained
• Both high and low abundant proteins can be detected
• More than one protein can be detected through multiplexing, permitting co-localization studies
• Subcellular particulate data (visualizing DNA or chromosomes) can be obtained more easily
   

• The antigenicity of the protein is better preserved
• Working with fresh tissues is non-toxic or non-carcinogenic since formalin is not used
• Processing time involved is considerably reduced with steps like epitope retrieval being eliminated
   

Tips and tricks

• Since OCT (optimal cutting temperature) compound has a tendency to autofluoresce, wash slides before fixing
• While washing the tissue section slides, extreme care should be taken to avoid peeling off of the sections
• To avoid the peeling-off of tissue sections from the slides, tissue can be sectioned on coated/charged slides
• To avoid cross contamination of samples on the same slide, use a PAP pen to create a hydrophobic barrier
• Since PAP pen marking also has a tendency to autofluoresce, clean slides before mounting without disturbing the stained tissue sections

Immunofluorescence analysis of sarcomeric alpha actin in mouse heart tissue using alpha actinin 2 Antibody (7H1L69), ABfinity Rabbit Monoclonal Antibody detected using goat anti-rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate.
 

Immunofluorescence analysis of corin in mouse heart tissue using alpha corin antibody, ABfinity Rabbit Oligoclonal Antibody, detected with goat anti-rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 conjugate.
 

Immunofluorescence analysis of cardiac troponin T in mouse heart tissue using cardiac troponin T monoclonal antibody, detected with goat anti-mouse IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 conjugate.
 

Search for the antibodies you need at thermofisher.com/antibodies
Learn more about IHC and how to get great images at thermofisher.com/ihc5steps

From sample preparation through image acquisition, Thermo Fisher Scientific offers a broad portfolio of superior reagents and instruments to help with every step of IHC workflow.

One of the critical reagents for a successful IHC experiment is the detection antibody. Thermo Fisher Scientific is striving to redefine the criteria of antibody performance by testing the specificity of antibodies in accordance with the newly adopted advanced verification testing methods for antibody validation. 

IHC analysis of p53 expression

Proteins can be expressed in some cell or tissue types but not in others. Antibody target verification can be determined by analyzing the relative expression of target proteins in different cell or tissue types by IHC. For example, the tumor suppressor p53 protein is known to be strongly expressed in the nuclei of tumor cells within ovarian cancer tissue compared to low expression seen in normal ovary tissue. Additionally, p53 expression is not detectable in other tissues, such as normal human liver. The immunohistochemistry (IHC) analysis of p53 expression in positive ovarian cancer and negative ovary and liver tissues shown in the figure demonstrates the specificity of the p53 antibody used for detection. As expected, p53 is highly expressed (stained brown) in the nuclei of ovarian cancer, but low in normal ovary tissue and not expressed in normal liver tissue.

Detection of relative p53 expression by IHC. The specificity of anti-p53 monoclonal antibody was demonstrated by detecting relative p53 expression in ovarian cancer (positive, left panel), normal ovary (low expression, middle panel) and normal human liver (negative, right panel) tissues. Tissues were deparaffinized with xylene, followed by rehydration in sequential washes of 100% ethanol, 95% ethanol, 80% ethanol, and water. To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0) and heated for 20 min in Lab Vision PT Module. Following antigen retrieval, tissues were blocked in a 10% goat serum in wash buffer solution for 30 minutes at room temperature and endogenous peroxidase activity quenched with Peroxidase Suppressor. Tissue was then probed with a p53 mouse monoclonal antibody at a concentration of 2.5µg/ml in 10% goat serum in wash buffer for 1 hour at room temperature in a humidified chamber. Tissues were washed extensively with PBST, and detection was performed using a SuperBoost goat anti-mouse Poly HRP secondary antibody reagent followed by chromogenic detection using DAB Quanto. Tissues were then counterstained with hematoxylin, mounted and imaged on EVOS FL Auto Imaging Station.
 

IF analysis of p53 expression

The same antibody verification and IHC workflow described above can be performed using immunofluorescence (IF) in place of the chromogenic detection. For fluorescence detection, the primary or secondary antibody is conjugated to a fluorophore that is detected by fluorescent microscope. Recently introduced, Alexa Fluor Plus secondary antibodies offer high signal-to-noise ratios and superior brightness. This figure depicts an example of IHC detection by IF using a p53 monoclonal primary antibody and an Alexa Fluor Plus 488 conjugated secondary antibody to visualize the relative expression of p53.

IHC detection of p53 across tissues by immunofluorescence. The specificity of anti-p53 monoclonal antibody was demonstrated by detecting relative p53 expression in ovarian cancer (positive, left panel), normal ovary (low expression, middle panel) and normal human liver (negative, right panel) tissues. Tissue sections were prepared, blocked and incubated with a p53 mouse monoclonal antibody at a concentration of 2.5µg/ml in 10% goat serum in wash buffer for 1 hour at room temperature in a humidified chamber. Tissues were washed extensively with PBST, and detection was performed using a goat anti-mouse IgG (H+L) Secondary Antibody, Alexa Fluor Plus 488 conjugate at a dilution of 1:100 for at least 30 minutes at room temperature in the dark (green). Nuclei (blue) were stained with Hoechst 33342. Images were taken on EVOS FL Auto Imaging Station.

Learn more about antibody verification

Get sharper and brighter images when using Invitrogen Alexa Fluor Plus secondary antibodies with new Invitrogen ProLong Glass Antifade Mountant. Alexa Fluor Plus secondary antibodies are highly cross-adsorbed and generate up to four times brighter signal than Invitrogen Alexa Fluor secondary antibodies when used in IF/ICC. ProLong Glass Antifade Mountant preserves the brighter signal from Alexa Fluor Plus secondary antibodies, minimizing signal loss due to photobleaching. When cells were exposed to constant illumination over 90 seconds, ProLong Glass reagent preserved over 90% of the signal from Alexa Fluor Plus secondary antibodies, illustrating its superior photobleaching protection. Meanwhile, ProLong Glass reagemt features low background in all fluorescence channels. Brighter signals combined with low background and better photobleaching protection yield higher signal-to-noise ratios, resulting in enhanced sensitivity required for detection of low-abundance targets.

Improved resolution with same-as-glass RI

ProLong Glass Antifade Mountant, which features improved refractive index of 1.52, increases axial resolution up to 75% and imageable focal depth by 3-4 times compared to mountants with refractive index of 1.47. ProLong Glass Antifade Mountant’s refractive index is same as slide glass, immersion oil and microscope objective, which minimizes optical refraction path and allows more light in microscope objective. Now, for the first time, researchers can take bright, sharp images of tissues and samples up to 150 µm thick, preserved with a hard-set mountant.

Alexa Fluor Plus secondary antibodies with Invitrogen ProLong Glass Antifade Mountant

Get the most from your samples and microscope by using Alexa Fluor Plus secondary antibodies with ProLong Glass Antifade Mountant. Alexa Fluor Plus secondary antibodies exhibit bright fluorophore technology, with the least background. ProLong Glass reagent preserves the bright signal and low background with antifade technology, and its near-perfect refractive index enhances focal depth and resolution of images. Bright and sharp images are possible when these two innovative technologies are used together.

Differentiated H9 NSC-derived neurons mounted with Prolong Glass Antifade Mountant, Alexa Fluor Plus secondary antibodies and imaged with EVOS FL Auto 2. H9 NSC-derived neurons after 3 weeks of differentiation in the Gibco B-27 Plus Neuronal Culture System, containing B-27 Plus Supplement and Neurobasal Plus Medium, and CultureOne Supplement. Cells were fixed and labeled with NucBlue Fixed Cell ReadyProbes Reagent, mouse anti-beta-3 Tubulin Antibody detected with Invitrogen Alexa Fluor Plus 488 Goat Anti-Mouse Secondary Antibody, ActinRed ReadyProbes Reagent, and rabbit anti-Synaptophysin Antibody (Cat. No. 18-0130) detected with Invitrogen Alexa Fluor Plus 647 Goat Anti-Rabbit Secondary Antibody (Cat. No. 32733). Cells were mounted in ProLong Glass Antifade Mounting Medium. Images acquired on EVOS FL Auto 2 using a 10x Olympus air objective.

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Features

• Speed—scan a 96-well plate in 3 fluorescent channels in less than 5 minutes
• Flexibility—customize the system with more than 20 user-changeable LED light cubes, dual cameras (monochrome and color), a variety of objectives ranging from 1.25x to 100x, and multiple vessel holders
• Time-lapse live-cell imaging—option for precise control of temperature, humidity, and gases for normoxic or hypoxic conditions allows a wide range of biological studies under physiological conditions with an onstage incubator
• Automation—time-saving features such as autofocus, rapid stage movement, and automated routines help reduce time to complete experiments, allowing high throughput, high data quality, and improved experimental reproducibility
• Data analysis—optional Celleste advanced software package for quantitative and statistical analysis

Learn more about our EVOS imaging solutions at thermofisher.com/microscopes

At a glance

The CellInsight CX7 LZR instrument offers:

• Faster scan times—take only seconds for confocal imaging of one picture
• Superior image quality—enhanced light performance illuminates the field evenly enabling image-quality quantitation
• Higher sensitivity at more wavelengths—expand your research with the capability to use a diverse array of dyes and multiplexing with no bleed through

Learn more about the CellInsight CX7 LZR

Learn more about the Invitrogen HCA On-Stage Incubator

GFP BrightComp eBeads Compensation Beads were designed for setting flow cytometry compensation when using green fluorescent protein (GFP).

• Consistent
• Accurate
• Simple-to-use reagent

With a near-identical spectral match to GFP at 3 levels of intensity, dispense the eBeads as a single drop for compensation made easy (see figures).

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Similar to Brilliant Violet 785 and Brilliant Violet 786, the new eBioscience Super Bright 780 dye expands your options for available violet-laser excitable antibody conjugates and joins the growing family of over 700 Super Bright antibody conjugates. Now available in five fluorophore formats (Super Bright 436, 600, 645, 702, and 780), view the Super Bright selection guide, see comparative data and select the Super Bright antibody conjugate for your flow cytometry panel.

Find out more at thermofisher.com/superbright

Fluorescence intensity and dot plot comparison of Super Bright 780 conjugates and Brilliant Violet 786 conjugates.(A) Mouse splenocytes stained with anti-CD4 conjugated to Super Bright 780(red) or Brilliant Violet 786 conjugate (gray), at the same concentration of antibody. (B) Human peripheral blood cells stained with anti-CD19 conjugated to (B) Brilliant Violet 786 or (C)Super Bright 780 at the same concentration of antibody. 

With this free virtual learning platform, you can access webinars, posters, and papers about applications and techniques related to protein gel chemistries, western detection, mass spectrometry, flow cytometry, fluorescence imaging, high-content imaging, antibodies, immunoassays, and other topics.

Learn more, visit thermofisher.com/proteincelledu

Representative titles include:

• Addressing the antibody reproducibility crisis: A panel discussion with key scientific leaders
• Understanding cell reprogramming in treatment-resistant prostate cancer using organoids
• Innovative quantitative imaging techniques and their utility in advancing neurobiology research
• Five steps for publication-quality fixed-cell imaging the first time
• What are the current advances in flow cytometry?

To access this and other webinars, visit themofisher.com/pcawebinars