Fitc conjugated anti mouse gl7

Antibodies for ATM (2873S; Cell Signaling; 1:1000), 53BP1 (NB100-304; NOVUSBIO; 1:1000), RIF1 (ab1213422; Abcam; 1:500), REV7 (A9861; Abclonal; 1:1000), REV1 (sc-393022; Santa Cruz; 1:1000), REV3L (GTX17515; Gene Tex; 1:1000), AID (A16217; Abclonal; 1:1000), MSH2 (ab227941; Abcam; 1:1000), β-actin (AC028; Abclonal; 1:10,000), FLAG (F1804, Sigma; 1:1000), β-Tubulin (A01030HRP; Abbkine; 1:10,000), glyceraldehyde 3-phosphate dehydrogenase (AB2000; Abways; 1:20,000), and Rabbit TrueBlot (18-8816-33; Ebioscience; 1:1000) were used in western blotting. PE-conjugated anti-mouse IgA (12-4204-83; Ebioscience; 1:200), APC-conjugated anti-mouse IgM (1020-11S; Southern biotech; 1:200), APC-conjugated anti-mouse B220 (553092, BD; 1:200), fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG1 (553443; BD; 1:200), FITC-conjugated anti-mouse IgG3 (553403; BD; 1:200), APC-eFluor780-conjugated anti-mouse B220 (47-0452-82, Invitrogen; 1:200), FITC-conjugated anti-mouse GL7 (144604, BioLegend; 1 : 200), PE-Cy7-conjugated anti-mouse CD95 (557653, BD; 1:200), and Fluorescein-labeled Peanut Agglutinin (FL-1071, Vector Laboratories; 1:500) were used in the fluorescence-activated cell sorting analysis.

Mediastinal lymph nodes were fixed with 4% paraformaldehyde fixative for 16 hours and then dehydrated in 30% sucrose overnight before being embedded in OCT (Tissue-Tek). OCT-embedded 10-mm cryostat sections were dehydrated in acetone prior to freezing. Sections were rehydrated in PBS and incubated with 1% SDS in PBS for 5 min. Sections were then blocked in PBS with 0.05% Tween-20 and 3% BSA for 1 hour prior to staining. Slides were stained with FITC conjugated anti-mouse GL-7 (diluted 1:100) and Alexa Fluor 647 conjugated anti-mouse IgD (1:200) (BioLegend) in 1%BSA in PBS/T overnight. Slides were washed and stained shortly with Hoechst (1:20,000) (Thermo Fisher Scientific). Sections were mounted with mounting medium (Sigma-Aldrich) and examined with a Zeiss LSM 880 confocal microscope. Imaging data were analyzed with Imaris 9.2 (Bitplane).