Visualization and Quantification of Germinal Centers

Spleens were flash-frozen in OCT compound. 6μm sections were cut on a cryostat and fixed in cold acetone as previously described (43 (link)). Spleen sections were stained with the following combination of antibodies: GL7-FITC, anti-CD4-PE (GK1.5), anti-IgD-APC (11–26c.2a) from BD Biosciences. Image acquisition was performed on a Leica DM4000 fluorescent microscope. The color intensity of images was consistently enhanced among images using Adobe Photoshop for better visualization, while maintaining the integrity of the data. For GC quantitation, GC size was measured on 10 randomly selected GCs (or the highest number available on the section) from 4 or more mice per group.

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Schell S.L., Bricker K.N., Fike A.J., Chodisetti S.B., Domeier P.P., Choi N.M., Fasnacht M.J., Luckenbill S.A., Ziegler S.F, & Rahman Z.S. (2021). Context-dependent miR-21 regulation of TLR7-mediated autoimmune and foreign antigen driven antibody-forming cell and germinal center responses. Journal of immunology (Baltimore, Md. : 1950), 206(12), 2803-2818.

Publication 2021

GL7-FITC DM4000 fluorescent microscope

Acetone Anti igd Antibodies Cold Fitc Frozen Mice Microscope Spleen

Corresponding Organization :

Other organizations : Pennsylvania State University

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Variable analysis

independent variables

Tissue processing (flash-freezing in OCT compound)
Antibody staining (GL7-FITC, anti-CD4-PE, anti-IgD-APC)

dependent variables

Germinal center (GC) size
Fluorescence intensity (color intensity of images)

control variables

Cryostat section thickness (6μm)
Fixation method (cold acetone)
Imaging platform (Leica DM4000 fluorescent microscope)
Image processing (Adobe Photoshop for color enhancement)

controls

Positive control: Not specified
Negative control: Not specified

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