RNAscope (Advanced Cell Diagnostics) in situ hybridization and immunofluorescence staining were performed on 4 μm FFPE sections from cystectomy specimens with existing scRNA-seq and TCR-seq data. Tissues were pre-treated with target retrieval reagents and protease to improve target recovery based on guidelines provided in the RNAscope Multiplex Fluorescent Reagent Kit v2 Assay protocol. Probes for human GZMB and GZMK mRNA (ACD) were incubated at 1:700 dilution for 2 hr 40 min. The probe was then hybridized with Opal 7-Color Manual IHC Kit (PerkinElmer), with detection of GZMB and GZMK using Opal 620 and Opal 540 respectively. Samples were then immunofluorescence stained for human CD4 (Cell Marque) which was detected using Alexa Fluor 555-conjugated goat anti-rabbit IgG secondary antibody (Invitrogen). Tissues were counterstained with DAPI. Slides were imaged using a TCS SP8 X white light laser inverted confocal microscope (Leica Microsystems). No staining was seen with negative control probes (for RNAscope) or with secondary antibody alone (for immunofluorescence) for tumor tissue from cystectomy specimens (shown in Figure 4F ) or healthy tonsil tissue (data not shown).
Tcs sp8 x white light laser inverted confocal microscope
Manufactured by Leica
The Leica TCS SP8 X white light laser inverted confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It features a tunable white light laser source that provides a continuous range of excitation wavelengths, allowing for flexible and efficient fluorescence imaging. The microscope's inverted design and confocal technology enable optical sectioning and high-resolution imaging of samples.
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2 protocols using tcs sp8 x white light laser inverted confocal microscope
1
Multiplexed Detection of GZMB and GZMK in Tumor Samples
2
Multiplexed Detection of Cytotoxic T-Cell Markers
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RNAscope (Advanced Cell Diagnostics) in situ hybridization and immunofluorescence staining were performed on 4 μm FFPE sections from cystectomy specimens with existing scRNA-seq and TCR-seq data. Tissues were pre-treated with target retrieval reagents and protease to improve target recovery based on guidelines provided in the RNAscope Multiplex Fluorescent Reagent Kit v2 Assay protocol. Probes for human GZMB and GZMK mRNA (ACD) were incubated at 1:700 dilution for 2 hr 40 min. The probe was then hybridized with Opal 7-Color Manual IHC Kit (PerkinElmer), with detection of GZMB and GZMK using Opal 620 and Opal 540 respectively. Samples were then immunofluorescence stained for human CD4 (Cell Marque) which was detected using Alexa Fluor 555-conjugated goat anti-rabbit IgG secondary antibody (Invitrogen). Tissues were counterstained with DAPI. Slides were imaged using a TCS SP8 X white light laser inverted confocal microscope (Leica Microsystems). No staining was seen with negative control probes (for RNAscope) or with secondary antibody alone (for immunofluorescence) for tumor tissue from cystectomy specimens (shown in Figure 4 F) or healthy tonsil tissue (data not shown).
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