Dextramer Optimization kit for in situ staining
Detection and localization of antigen-specific T cells in tissues has proven difficult. The affinity and number of detectable TCRs varies with choice of antigen, tissue, preservation method and fixation. For the optimization of in situ staining Immudex offers a kit comprising any 3 Dextramers of the same specificity but Low, Medium or High avidity. The different avidities result from different numbers of MHC-peptide complexes carried by the 3 Dextramers.
The FITC-conjugated Dextramers of the Optimization Kit carry a large number of FITC molecules per reagent. The FITC molecule may be used directly as a fluorescent label (see figure 2), or as a tag for a secondary antibody/visualization system (see figure 1). A Guideline protocol for in situ staining using Dextramers is provided below.
In the following in situ staining experiment a frozen tissue section from a mouse spleen was analyzed, using the Guideline protocol provided below:
Figure 1: In situ staining of transgenic mouse spleen. A mouse spleen section was stained with positive control (left) or negative control (right) FITC-conjugated Dextramer, and visualized using rabbit anti-FITC/Envision anti-rabbit HRP (Dako P5100/K4003). Left panel: H-2Db(KAVYNFATC)/FITC Dextramer; Right panel: H-2Db(FQPQNGQFI)/FITC Dextramer.
In the following example the FITC-conjugated Dextramer was detected by the FITC-fluorescence directly, i.e. without the use of amplification/visualization systems:
Figure 2: Detection of Survivin-specific cytotoxic T cells in biopsies of metastatic lesions from a melanoma patient. Cryopreserved sections were dried, fixed in acetone, incubated with TRITC-labeled anti-CD8 antibody followed by incubation with FITC-conjugated HLA-A2(survivin) Dextramer, and finally nuclei counter-stained with DAPI. A) stained nuclei, B) stained CD8+ cells, C) stained survivin-specific T cells, and D) an overlay of A, B and C. The data was kindly provided by Professor Jürgen Becker, Medical University Graz (for protocol, see publication no. 8 in Publication list hereunder).
- Guideline for in situ staining using Dextramers
- Publications on in situ staining using MHC Dextramers
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