In the preclinical development pipeline, test items are often applied in vivo by systemic injections. In order to prove that the test item—such as therapeutic antibodies, peptides, proteins, or antibody-drug conjugates (ADC)—is able to cross the blood brain barrier, to enter the brain parenchyma, and to label its intended target, the test item needs to be detected and visualized. This is where tags become useful tools for easy detection of test items. The histology department at Scantox Neuro has a lot of experience in applying tagged detection strategies. Commonly used tags include:
• Biotin
• Hexahistidine (HIS)
• Hemagglutinin (HA)
Peptides and proteins are often fused to fluorescent proteins such as green fluorescent protein (GFP) and red fluorescent protein (RFP), and their signal can be detected either directly by their epifluorescence or indirectly using antibodies if the signal is too weak or even absent because of the use of heat-induced antigen retrieval.
Human, or humanized antibodies, increasingly used as test items and often systemically applied, can be detected by anti-human secondary antibodies. If the signal is weak, different ways of antigen retrieval may be helpful, and enzyme-based signal amplification can enhance visibility.
Figure: A: Virally expressed protein fused to GFP; pyramidal cells at hippocampal CA2/3 show intense GFP signal detected by anti-GFP antibody (green); Iba1 (red). B: Biotinylated test item was injected intravenously and binds to amyloid plaques in 5xFAD mice; biotin was detected by Streptavidin-HRP and Aluora signal amplification (red); CD11b (white), Synaptophysin (pink), NeuN (green). C: Systemically applied test item preferentially localized in cerebellar Purkinje cells; HA tag was detected by anti-HA antibody followed by fluorophore-conjugated secondary antibody (red). D: Plaque decoration in mouse brain subiculum by systemically applied human antibody targeting beta-amyloid; test item was detected by fluorophore-conjugated anti-human antibody (red); Iba1 (turquoise), amyloid (MOAB-2 antibody, green). A-D: Nuclei were stained by DAPI (blue).
Scantox Neuro routinely performs multiplex fluorescence labeling, combining tag detection with additional markers (e.g., neuronal, glial, or vascular markers) and DAPI staining for nuclear visualization. This enables both qualitative and quantitative assessment of test item distribution, cellular localization, and target engagement.
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