Immunohistochemical Analysis of Splenic and Renal Tissues

Splenic and kidney sections were embedded in Tissue-Tek OCT compound (Sakura Finetek) and rapidly frozen in a freezing bath of dry ice and 2-methylbutane. Frozen OCT samples were cryosectioned and unstained slides were stored at −80°C. Immunohistochemical staining procedures were performed as previously described (1 (link), 2 (link), 5 (link)). For detection of germinal centers in the spleen, the following monoclonal Abs were used: GL7-AF488, IgD-PE, and CD4-allophycocyanin (all from BioLegend). For detection of renal deposition, C3-FITC (Cedarlane, Burlington, NC) and IgG2a-PE (eBioscience) Abs were used. Images were captured with a Zeiss LSM 880 confocal microscope (Fralin Imaging Center, Virginia Tech). Corrected total cell fluorescence scores were calculated with ImageJ software (National Institutes of Health, Rockville, MD).

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Cabana-Puig X., Bond J.M., Wang Z., Dai R., Lu R., Lin A., Oakes V., Rizzo A., Swartwout B., Abdelhamid L., Mao J., Prakash M., Sangmeister C., Cheung N., Cowan C., Reilly C.M., Sun S., Ahmed S.A, & Luo X.M. (2022). Phenotypic Drift in Lupus-Prone MRL/lpr Mice: Potential Roles of MicroRNAs and Gut Microbiota. ImmunoHorizons, 6(1), 36-46.

Publication 2022

Tissue-Tek OCT compound GL7-AF488 IgD-PE CD4-allophycocyanin IgG2a-PE

Corresponding Organization :

Other organizations : Virginia Tech, Virginia–Maryland College of Veterinary Medicine, Carilion Roanoke Memorial Hospital, Carilion Clinic, Edward Via College of Osteopathic Medicine, University of California, Irvine

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Variable analysis

independent variables

Tissue type (splenic and kidney sections)

dependent variables

Immunohistochemical staining patterns for germinal centers in the spleen and renal deposition
Corrected total cell fluorescence scores

control variables

Embedding of tissue sections in Tissue-Tek OCT compound
Rapid freezing of OCT samples in a freezing bath of dry ice and 2-methylbutane
Storage of unstained slides at −80°C
Immunohistochemical staining procedures used in previous studies (1, 2, 5)

positive controls

None specified

negative controls

None specified

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