Immunofluorescence Microscopy

Fluorescently Conjugated Antibodies For Imaging

Immunofluorescence microscopy relies on fluorescently labeled antibodies for target detection in single cells or tissue sections with a high spatial resolution. Randomly labeled antibodies, often used for their convenience and availability, compromise the quality of the conjugate resulting in high noise-levels without quantitation possibilities. The GlyCLICK technology for site-specific fluorescence labeling of native IgG produces conjugates that improve the imaging quality.

 

GlyCLICK workflow - fluorescence labeling

Site-Specific Fluorescence Labeling

Intricate imaging experiments such as multiplexed designs often require the use of different target-specific antibodies labeled with fluorophores that have minimal spectral overlap. Ensuring the same level of labeling for each conjugate is central in order to minimize the risk of introducing artifacts that may impact the imaging analysis. To evaluate the quality of the conjugation using the GlyCLICK technology, trastuzumab was labeled with AlexaFlour®488 and digested into intact Fc/2 and F(ab’)2 subunits using FabRICATOR prior to RP-HPLC analysis. The resulting peaks show site-specific labeling only at the Fc/2 subunit leaving the antigen-binding F(ab’)2 region unaffected and preserved (Fig. 1).

GlyCLICK World ADC 2019 Poster

fluorescence labeling - Glyclick

Figure 1.  RP-HPLC analysis of trastuzumab performed on an Agilent 1290 using Waters Acquity UPLC® BEH C4, 1.7 um, 2.1 mm column in an acetonitrile/ isopropanol gradient at 65° C.

Reliable Antigen Detection with GlyCLICK

 

Immunostaining using randomly labeled antibodies can influence the image quality through inadequate signals and unspecific binding due to excessive labeling or impaired immunoreactivity from labels resided in the antigen-binding Fab region. To evaluate the immunoreactivity after labeling, trastuzumab was site-specifically conjugated to AlexaFluor® 488 using GlyCLICK or by random labeling at lysines using the DyLight®488 kit. The resulting SPR response curves show that only 20% of the randomly labeled material retained binding capacity while GlyCLICK conjugated antibodies display full preserved immunoreactivity for sensitive and reliable detection (Fig. 2).

 

Figure 2. SPR analysis. Anti-human IgG (Fc) captured trastuzumab: native, DyLight488 conjugated or GlyCLICK conjugated to AlexaFluor®488 (DOL=2). HER2 was injected in a range to ensure sufficient curvature. All data were fitted against a 1:1 mathematical model.

Optimal Intensities for Dynamic Imaging

Dynamic fluorescence labeling

 

Immunofluorescence microscopy requires not only reliable antigen-binding, but the ability to obtain optimal intensity levels for optical analysis without saturation or loss of signals for high or low antigen expression. The quality of a microscopy analysis using GlyCLICK conjugated trastuzumab with AlexaFluor®647 was studied by imaging of the HER2 expression on cells and in paraffin-embedded tissue. The images show a dynamic visualization of the antigen distribution and expression levels with minimal noise levels or unspecific staining at optimal intensity levels for minimal saturation or loss of signal (Fig. 3).

 

Figure 3. Confocal microscopy of MDA-MB-453 PFA-fixed cells or paraffin-embedded (BSA) blocked tissue. Left: cells with DAPI (0.2uM) and T-GlyCLICK-AlexaFluor®647 (1ug/ml), Middle: cells with DAPI (0.2 uM), phallodinAF488, T-GlyCLICK-AlexaFluor®647 (5ug/ml), Right: tissue with DAPI (0.2uM), T-GlyCLICK -AlexaFluor®647 (10ug/ml).

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