Fluorescent images were taken with a Zeiss LSM700 Confocal Microscope. All images are Maximum intensity projections. Images of samples that did not fit the microscope field of view were obtained using the tile scan function (Zeiss ZEN) that aligns and stitches tiles to obtain a single image of a large specimen. Images were processed using ImageJ (Fiji) (Schindelin et al., 2012 (link)). Light images were taken with a Zeiss Discovery Microscope. Cell counting was performed manually after blinding control and experimental conditions.
Tile scan function
Manufactured by Zeiss
The Tile scan function is a feature available in select Zeiss lab equipment. It enables the capture of larger samples or areas by stitching together multiple individual images, producing a comprehensive high-resolution overview. The core function of this feature is to facilitate the acquisition of detailed data from extended specimens or regions of interest.
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2 protocols using tile scan function
1
Confocal and Light Microscopy Imaging
2
Multicolor Lymph Node Imaging
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Lymph nodes were embedded in TissueTek OCT compound and 8 µm cryostat sections were prepared and fixed in acetone. After peroxidase inactivation and blocking of unspecific binding sites with Casein Solution (Vector Laboratories) supplemented with mAb 2.4G2 (100 µg/ml), sections were stained with FITC-coupled OX-7 (anti-Thy-1.1), Alexa Fluor 555–coupled RA3-6B2 (anti-B220), and Alexa Fluor 594–coupled GK1.5 (anti-CD4). Signals for Thy-1.1 were amplified with peroxidase-conjugated anti-FITC followed by Alexa Fluor 647–coupled tyramide (Invitrogen). Nuclei were stained with DAPI. Whole lymph node sections were captured on an LSM 780 with ZEN2010 imaging software using the tile-scan function (Carl Zeiss).
At least 6 sections from different areas of each lymph node were used for quantification of signals. For this purpose, total lymph node area (any signal) and B cell follicles (B220+) were defined and transgenic T cell signal (Thy-1.1) was separated from background and unified by setting an appropriate threshold. Localization of T cells was determined by automatically counting pixels positive for the Thy-1.1 signal in the distinct lymph node compartments. Mean B cell follicle sizes were similar between groups.
At least 6 sections from different areas of each lymph node were used for quantification of signals. For this purpose, total lymph node area (any signal) and B cell follicles (B220+) were defined and transgenic T cell signal (Thy-1.1) was separated from background and unified by setting an appropriate threshold. Localization of T cells was determined by automatically counting pixels positive for the Thy-1.1 signal in the distinct lymph node compartments. Mean B cell follicle sizes were similar between groups.
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