Rat anti mouse b220

To examine GC architecture, spleens were removed from mice and fixed in 10% formalin (Sigma-Aldrich). For immunostaining, paraffin sections where deparaffined, hydrated and treated with antigen retrieval buffer (Dako). The sections then were permeabilized by 4 min treatment with Triton X-100 (0.1% in PBS) and incubated for 90 min with blocking buffer (Dako) followed by an overnight incubation with primary antibodies: rat anti-mouse B220 and hamster anti-mouse CD3e (1/1000, BD Pharmingen). For immunohistochemistry, antibodies were detected with AP-conjugated goat anti-Armenian hamster IgG or HRP-conjugated donkey anti-rat IgG (1/10000, Jackson ImmunoResearch Laboratories). HRP was reacted with DAB (Peroxidase Substrate Kit; Vector), and alkaline phosphatase with Fast Blue/Napthol AS-MX (Sigma-Aldrich). Levamisole (Sigma) was used to block endogenous alkaline phosphatase activity and slides were mounted in Crystal Mount (Electron Microscopy Sciences). Sections were viewed under a Nikon Microphot FXA light microscope and photographs were taken with a Spot Insight Camera, using 10× and 40× objectives, then analyzed using Spot Advanced software (Diagnostic Instruments) and ImageJ (NIH).

Brain tissue fixed in paraformaldehyde was embedded in paraffin and coronally sectioned for immunofluorescent staining. All sections were deparaffinized and rehydrated, heated with either citrate buffer (pH 6) or Tris-EDTA buffer (pH 9) for antigen retrieval, and then blocked with 20% normal horse serum. Depending on the stain, sections were incubated with primary antibodies of:
(1) rat anti-mouse B220 (1:100; BD, Franklin Lakes, NJ) and rabbit anti-mouse CD3e (1:100; Invitrogen, Waltham, MA), followed by the secondary antibodies Alexa Fluor 488-conjugated donkey anti-rat and Cy5-conjugated donkey anti-rabbit (both 1:100), respectively;
(2) rabbit anti-mouse syndecan-1 (SDC1) (1:100; Sino Biological, Wayne, PA) and Alexa Fluor 594-conjugated donkey anti-mouse IgG (1:300), followed by the secondary antibodies Alexa Fluor 488-conjugated donkey anti-rabbit (1:100) and Alexa Fluor 594-conjugated donkey anti-mouse IgG (1:500), respectively;
(3) rat anti-human/mouse activation-induced cytidine deaminase (AID) (1:50; Invitrogen, Waltham, MA), followed by biotin-conjugated donkey anti-rat and amplified by the Tyramine Signal Amplification (TSA) Cyanine 5 System from Perkin Elmer (Waltham, MA).
All fluorophore-conjugated antibodies were from Jackson ImmunoResearch (West Grove, PA).