Pna fluorescein

Spleens were embedded and frozen in OCT compound (Electron Microscopy Sciences) prior to sectioning. Four micron splenic sections were then fixed in 75% ethanol/25% methanol for 10 mins at −20°C. After blocking, primary Abs CD3e and IgD (eBioscience) were applied for 4 hours at 25°C or overnight at 4°C. PNA-Fluorescein (Vector Labs), anti-rat DyLight649, and anti-hamster Cy3 (Jackson ImmunoResearch) secondary Abs were then applied for 1 hour at 25°C. Slides were mounted with Fluoroshield (Electron Microscopy Sciences) and subsequently imaged on a Cannon Ti-U fluorescent microscope. Images were acquired with a D5-QilMc digital camera in conjunction with NIS Elements software. For GC counts, staining was performed as above and images were captured on an EVOS FL Auto microscope (Life Technologies).

Kidneys frozen in liquid nitrogen were embedded in Optimal Cutting Temperature compound (Ted Pella Inc.). Tissue was cryosectioned (5 μm) and permeabilized with cold acetone for 20 min at room temperature. The slides were washed with PBS and blocked with 2% horse serum prepared in 3% BSA/PBS for 20 min at room temperature. The sections were stained for 30 min with 1:200 diluted horse anti-mouse IgG-Fluorescein (Vector Laboratories) followed by extensive washing in PBS containing 0.1% Tween 20 and mounting using ProLong Diamond Antifade (Thermo Fisher Scientific) containing DAPI counterstain. Images were acquired using Axiovert 200 microscopy system with Apotome imaging and Axiovision software (Carl Zeiss Microscopy, LLC). For GC studies, spleen sections were rehydrated for 30 min with PBS and blocked for 1 h with 10% BSA/PBS at room temperature. The sections were stained for 1 h with 1:100 diluted PNA-Fluorescein (Vector Laboratories) or anti-GL7-Alexafluor 488 and washed three times with PBS, followed by incubation with 1:50 diluted PE-anti-mouse CD19 antibody (Thermo Fisher Scientific) at room temperature for 1 h. The slides were washed and mounted using ProLong Diamond Antifade (Thermo Fisher Scientific) containing DAPI. Germinal centers were mapped using the selection tool and quantified using ImageJ (NIH). H & E stained spleen sections were also imaged for GC analyses.