Ihc icc blocking high protein buffer

Spleens were placed in Tissue-Tek optimum cutting temperature compound (Sakura Finetek), snap-frozen in liquid N₂ and stored at −80° C until sectioned by HistoServ (Gaithersburg, MD). Affixed cryostats sections (6μm in thickness) were dried at 25° C, fixed in ice-cold acetone for 10 min and dried again at 25° C. Slides were rehydrated in 1x Tris-buffered saline (TBS) pH 7.6, place in a humidifier chamber. Sections were blocked for 2h at 25° C with IHC/ICC Blocking High Protein Buffer (eBioscience). Sections were stained overnight at 4° C in blocking buffer with anti-IgD (11–26c) PerCP e-Fluor 710 (eBioscience), anti-CD4 (GK1.5) Alexa Fluor 647 (Biolegend), anti-GL7 Alexa Fluor 488 (Biolegend). Slides were then washed in 1x TBS for 5 min with gentle agitation. Slides were mounted with Fluoromount-G with DAPI (eBioscience). Images were visualized and collected using a Leica SP8 inverted 5-channel confocal microscope (Leica) and analyzed in Imaris 8.1 (Bitplane, Oxford Instruments).

H & E staining of sections of spleen, salivary glands, kidneys, lung, small intestine and liver from Eos^fl/fl and Eos cKO mice were performed by HistoServ Labs Inc. (Gaithersburg, MD). Confocal images were taken in the Biological Imaging Section, NIAID, NIH. Histology scores were determined by an independent scorer.
Affixed cryostat sections were dried at 25 °C, fixed in ice-cold acetone for 10 min and dried again at 25 °C. Slides were rehydrated with 1x Tris-buffered saline (TBS) pH 7.6 and placed in a humidifier chamber. Sections were blocked for 2 h at 25 °C with IHC/ICC Blocking High Protein Buffer (eBioscience). Sections were stained overnight at 4 °C in blocking buffer with anti-IgD PerCP e-Fluor 710 (11–26c; eBioscience), anti-CD4 Alexa Fluor 647 (GK1.5; Biolegend), anti-GL7 PE (Biolegend). Slides were then washed in 1x TBS for 5 min with gentle agitation. Slides were mounted with Fluoromount-G with DAPI (eBioscience). Images were visualized and collected using a Leica SP8 inverted 5-channel confocal microscope (Leica) and analyzed in Imaris 8.1 (Bitplane, Oxford Instruments). Images were taken in the Biological Imaging core, NIAID, NIH.