Anti igg biotin

Mouse kidneys and spleens were removed postmortem at 36 weeks of age and embedded in OCT compound (Sakura Finetek, Torrance, CA, USA) and snap frozen in liquid nitrogen. Frozen sections were fixed in acetone (Sigma-Aldrich), blocked with 10% normal donkey serum (Sigma-Aldrich), and stained with 1:200 dilution of anti-IgG-biotin (Sigma-Aldrich) and 1:200 dilution of streptavidin-Cy3 (Invitrogen, Grand Island, NY, USA), 1:200 dilution of anti-C3-FITC (ICN Biomedicals, Irvine, CA, USA), 2 µg/ml anti-B220-allophycocyanin (eBioscience), 5 µg/ml anti-GL7-FITC (BD Biosciences), 1 µg/ ml anti-CD4-Alexa Flour 647, and 1 µg/ml anti-B220-Alexa Flour 488 (Caltag Laboratories, Buckingham, MK, UK). Fluorescence images were acquired using a TCS SP5 confocal microscope (Leica, Ernst-Leitz-Strasse, Wetzlar, Germany). The area of GCs per field of 200× magnification was calculated using ImageJ software (NIH, Bethesda, MD, USA). GCs were classified as small (<7,000 µm 2 ), medium (7,000-14,000 µm 2 ), and large (>14,000 µm 2 ).