Immunofluorescent Staining of Spleen

Spleens were prepared for staining as previously described (34 (link)). Slides were stained with a primary antibody cocktail of CD169-FITC (MOMA-1; MCA947F; Bio Rad Laboratories; diluted 1:50), IgM-Biotin (II/41; 553436; BD Biosciences; diluted 1:35), and CD1d (1B1; 123551; BioLegend; diluted 1:200) in serum blocking buffer. Slides were secondary stained with anti-FITC-A488 (polyclonal; A11090; Life Technologies; diluted 1:100) and streptavidin-Hilyte555 (60666; Anaspec; diluted 1:20) in serum blocking buffer. Slides were imaged on a Zeiss Axio Observer Z1 at room temperature with acquisition software Zen v2.0.0.0 (Blue Edition) using tiling and z-stacking. Images were taken with a 20X Plan-Apochromat 20x/0.8 objective (420650–9901; Zeiss) and an Axiocam 506 mono digital camera (Zeiss). Image files were analyzed with ImageJ v1.52k (NIH).

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Haines R.R., Scharer C.D., Lobby J.L, & Boss J.M. (2019). LSD1 cooperates with non-canonical NF-κB signaling to regulate marginal zone B cell development. Journal of immunology (Baltimore, Md. : 1950), 203(7), 1867-1881.

Publication 2019

Axio Observer Z1 20X Plan-Apochromat 20x/0.8 objective Axiocam 506 mono digital camera

Antibody cocktail Biotin Buffer Camera Cd1d Fitc Serum Streptavidin

Corresponding Organization : Emory University

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Variable analysis

independent variables

Primary antibody cocktail of CD169-FITC, IgM-Biotin, and CD1d

dependent variables

Staining patterns observed on the slides

control variables

Slides
Serum blocking buffer
Staining protocol
Imaging conditions (Zeiss Axio Observer Z1, 20X Plan-Apochromat 20x/0.8 objective, Axiocam 506 mono digital camera)

controls

None explicitly mentioned

Annotations

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