Anti mouse ig fitc

Three μm thick sections were cut with a cryostat from fresh-frozen samples. They were collected on positively charged slides and fixed for 10 minutes in cold acetone. For direct immunofluorescence, sections were incubated with the following FITC-conjugated antibodies: anti-human Kappa Light Chain-FITC (Diagnostic BioSystems, Pleasanton, CA, USA), anti-human Lambda Light Chain-FITC (Diagnostic BioSystems), anti-human Kappa Light Chain-FITC (Cytognos, Salamanca, Spain), and anti-human Lambda Light Chain-FITC (Cytognos). For indirect immunofluorescence, sections were first incubated with the following primary antibodies: anti-human Amyloid A (mc1 clone, Dako Agilent, Santa Clara, USA), and anti-human P Component (Dako). Sections were then incubated accordingly, with the appropriate secondary FITC-conjugated antibodies: anti-rabbit IgG-FITC (Southern Biotech, Birmingham, USA), and anti-mouse Ig-FITC (Southern Biotech).

Before sacrifice, three animals from each group were infused i.v. with 10 ml of 3.24 mg/ml purified CD3 Ab (clone PPT3) and sacrificed 10 min later. Lymphocytes were isolated and stained ex vivo with anti-mouse Ig-FITC (SouthernBiotech), which labels the circulating intravascular cells. The cells are washed, and normal mouse serum is then added to block any remaining binding capacity of the anti–Ig-FITC. The cells are then washed again and incubated with CD3 Ab labeled with PeCy5 (Abcam). This will bind unsaturated sites of the circulating cells, which are therefore double labeled, as well as all the sites on the T_RM that are not accessible to the CD3 Ab, given i.v. T_RM is therefore single labeled with PeCy5.
To allow intracytoplasmic staining of T_RM, the i.v. CD3 was detected with goat anti-mouse IgG BV421 (BioLegend) and blocked using normal mouse serum as above. Surface markers used were CD3ε-biotin PPT3 (Abcam), CD4 clone 74-12-4 PerCpCy5.5, CD8α-FITC 76-2-11 (all BD Biosciences), and Near-Infrared Fixable LIVE/DEAD stain (Invitrogen). Biotinylated CD3 was visualized with a streptavidin AF647 (BioLegend). Cells were permeabilized using Cytofix/Cytoperm before intracellular staining with IFN-γ PE P2G10 (BD Biosciences) and cross-reactive anti-human TNF-α-AF650 Mab11 (BioLegend). Samples were fixed in 1% paraformaldehyde before analysis using an LSRFortessa.