Anti-nuclear Immunofluorescence of Lyn-/- Mice

Anti-nuclear immunofluorescence was performed as previously described(23 (link)). Briefly, Kallestad HEp-2 slides (BIO-RAD) were stained overnight with 1µL of serum from either Lyn^−/−IgD^+/− or wildtype mice. After washing, antibody binding was detected using an anti-mouse secondary antibody conjugated to Alexa488 dye (Thermo) and DAPI counterstain (Thermo). Cells were imaged with the Crest LFOV Spinning Disk/ C2 Confocal at 1000x magnification under identical camera exposure and laser settings for knockout and control mice. Micrograph exposure was normalized to secondary only negative control in FIJI and applied to all images at once.

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Rackaityte E., Proekt I., Miller H.S., Ramesh A., Brooks J.F., Kung A.F., Mandel-Brehm C., Yu D., Zamecnik C., Bair R., Vazquez S.E., Sunshine S., Abram C.L., Lowell C.A., Rizzuto G., Wilson M.R., Zikherman J., Anderson M.S, & DeRisi J.L. (2023). Validation of a murine proteome-wide phage display library for the identification of autoantibody specificities. bioRxiv.

Publication Preprint 2023

HEp-2 slides Alexa488 dye DAPI counterstain

Anti antibody Antibody Cells Crest Dapi Immunofluorescence Mice Serum

Corresponding Organization : Chan Zuckerberg Initiative (United States)

Other organizations : Memorial Sloan Kettering Cancer Center

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Variable analysis

independent variables

Genotype: Lyn-/- IgD+/- mice vs. wildtype mice

dependent variables

Anti-nuclear immunofluorescence staining pattern

control variables

Staining procedure: Overnight incubation with 1µL of serum, washing, detection with anti-mouse secondary antibody conjugated to Alexa488 and DAPI counterstain
Imaging: Crest LFOV Spinning Disk/ C2 Confocal at 1000x magnification, identical camera exposure and laser settings for knockout and control mice
Image normalization: Normalized to secondary only negative control in FIJI and applied to all images

controls

Positive control: Not explicitly mentioned
Negative control: Secondary only (anti-mouse secondary antibody conjugated to Alexa488 and DAPI counterstain)

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