Mice were sacrificed and perfused with ice-cold PBS. Spleens were harvested and snap frozen in OCT tissue-freezing solution and stored at -80°C. 8μm sections were cut, mounted to slides, and fixed with ice-cold acetone. Immediately prior to performing immunohistochemistry, sections were rehydrated and then blocked with 5% FCS. Reagents used to stain GCs were: IgD (PE, clone 11-26c.2a, Biolegend, 1:100 dilution) and PNA (fluorescein labeled, Vector Labs, 1:200 dilution). Hoechst (1:2,500 dilution) staining was then performed and slides were mounted with Fluoromount G. Fluorescent images were taken of the entire spleen sections using a fully automated, brightfield/fluorescence slide scanning system (AxioScan.Z1, ZEISS). Spleens were imaged at 40X. Images were stitched in Zen Software (Blue Edition) v3.1 (ZEISS) and then uploaded to Imaris Software v8.4 (Bitplane) for quantification of GCs, which were manually counted for each image and defined as dense areas (>50μm in diameter) of PNA staining within IgD+ follicles. Analysis was performed blinded, and two sections were averaged for each individual spleen value. Tcra−/− mice that received no T cell transfers but were immunized with NP-LLOLT-N were used as controls.
Zen software blue edition v3
Manufactured by Zeiss
The Zen Software (Blue Edition) v3.1 is a microscope control and image processing software developed by Zeiss. It provides a user interface for controlling Zeiss microscopes and managing the acquired images. The software supports various microscopy techniques and offers basic image processing capabilities.
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2 protocols using zen software blue edition v3
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Quantification of Germinal Centers in Mice
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Quantification of Germinal Centers in Mice
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Mice were sacrificed and perfused with ice-cold PBS. Spleens were harvested and snap frozen in OCT tissue-freezing solution and stored at -80°C. 8μm sections were cut, mounted to slides, and fixed with ice-cold acetone. Immediately prior to performing immunohistochemistry, sections were rehydrated and then blocked with 5% FCS. Reagents used to stain GCs were: IgD (PE, clone 11-26c.2a, Biolegend, 1:100 dilution) and PNA (fluorescein labeled, Vector Labs, 1:200 dilution). Hoechst (1:2,500 dilution) staining was then performed and slides were mounted with Fluoromount G. Fluorescent images were taken of the entire spleen sections using a fully automated, brightfield/fluorescence slide scanning system (AxioScan.Z1, ZEISS). Spleens were imaged at 40X. Images were stitched in Zen Software (Blue Edition) v3.1 (ZEISS) and then uploaded to Imaris Software v8.4 (Bitplane) for quantification of GCs, which were manually counted for each image and defined as dense areas (>50μm in diameter) of PNA staining within IgD+ follicles. Analysis was performed blinded, and two sections were averaged for each individual spleen value. Tcra−/− mice that received no T cell transfers but were immunized with NP-LLOLT-N were used as controls.
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