Tsa cy3 system

H&E and immune histological staining of colonic sections with Abs directed against CD3 and Foxp3 were carried out as described previously 52 . Immunofluorescence of cryo-sections was performed using the TSA Cy3 system (PerkinElmer), a fluorescence microscope (IX70;
Olympus) and primary Abs against CD11c (HL3, BD Biosciences), MPO (15484, Abcam), and F4/80 (BM8, eBioscience). In brief, cryo-sections were fixed in ice-cold acetone for 10 minutes followed by sequential incubation with methanol, avidin/biotin (Vector Laboratories), and protein blocking reagent (Dako). Slides were then incubated overnight with primary Abs.
Subsequently, the slides were incubated for 30 minutes at room temperature (RT) with biotinylated secondary Abs (Dianova). All samples were finally treated with streptavidinhorseradish peroxidase and stained with tyramide (Cy3) according to the manufacturer's instructions (Perkin Elmer). Before examination, nuclei were counterstained with Hoechst 3342 (Invitrogen). Histological staining of splenic and thymic paraffin sections were carried out using Abs directed against CD3 (1:100, Serotec, MCA1477), B220

System; PerkinElmer) for 1 hat RT. The cells were incubated with anti-digoxigenin-POD (1 : 200 in TNB, Roche) and rabbit anti-Phospho-S6 Ribosomal Protein antibody (1 : 400 in TNB, #4858S, Cell Signaling) in a humidified chamber overnight in 4°C, next they were washed with PBS-T, and incubated with Alexa 488-conjugated anti-rabbit IgG secondary antibody (1 : 1000; Invitrogen). The hybridization signal was amplified with TSA Cy3 System (Perkin Elmer). The imaging was performed using confocal microscope LSM 700, Axio Imager Z2 (Zeiss) in a stack of 0.4 μm thick Z section, using a 40× objective (oil immersion; 1.3 NA) with 1.2 zoom factor. The obtained stack was "flattened" into a single image using a maximum intensity projection. Quantification of fluorescence signal intensity was performed in ImageJ software. Signal intensity was averaged on the cell area.