Alexa fluor 488 anti mouse igg

Mouse spleens were collected at 7 days post immunization, embedded in OCT medium and frozen at −80 °C before processing into 5 μm sections. Samples were fixed in cold 4% paraformaldehyde for 10 min followed by blocking in 5% FBS for 1 h at room temperature. For GC analysis, Alexa-Fluor 594 B220 (Biolegend 103254), Alexa-Fluor 488 anti-mouse IgG (Biolegend 405319), and Alexa-Fluor 647 anti-GL-7 (Biolegend 144605) or for macrophage depletion, Alexa-Fluor 594 B220, FITC anti-mouse CD68 (Biolegend 137005) /FITC anti-mouse CD11c (Biolegend 117305), and Alexa-Fluor 647 anti-mouse CD169 (Biolegend 142407) were incubated at 1:100 overnight at 4 °C. Samples were mounted on glass slides in ProLong Antifade reagent (Invitrogen). Images were acquired on a Zeiss LSM700 confocal microscope and on a Mirax Midi slide scanner system for whole organ imaging. GCs were defined as discrete areas of B220, GL-7, and IgG signal colocalization and counted manually in each spleen section. PBs were defined as IgG⁺ cells and quantified in two 1 mm² areas in each spleen section using ImageJ.

Expression of iTS and aTS recombinant proteins by trypomastigotes were analyzed by confocal microscopy. Approximately 10⁶ parasites were washed with PBS supplemented with 1% BSA. Parasites were incubated with rabbit IgG anti-FLAG (Sigma, diluted 1:300) to detect recombinant proteins and the mouse monoclonal antibody (mAb) 13G9 anti-TS (1:100) (Buschiazzo et al., 2002 (link)), to detect all trans-sialidases either endogenous or recombinant. Parasites were labeled with secondary AlexaFluor568-antibodies anti-rabbit IgG and AlexaFluor488-anti-mouse IgG (diluted 1:1,000, both from Biolegend). Fluorescence imaging was carried out under an Olympus FV1000 microscope equipped with a Plan APO N 60x oil, 1.42 NA, FN 26.5 objective.

CD4 T cells were treated with Fix Buffer I (BD Biosciences), Perm Buffer III (BD Biosciences), and stained with anti-human antibodies as follows: FITC anti-CD4 (BioLegend, Clone OKT4), PE-Cy7 anti-Bcl-6 (BioLegend, Clone 7D1), PE-anti-IL-21 (BioLegend, Clone 3A3-N2), Purified anti-AMPKα Antibody (BioLegend, Clone 5C9-5B10-6D3) plus Alexa Fluor^® 647 anti-mouse IgG (Thermo Fisher Scientific, Cat. A-21235), Alexa Fluor^® 647 mouse anti-human leptin receptor (BD Biosciences, Cat. 564376), Purified anti-AMPKα Phospho (Thr172) Antibody (BioLegend, Clone A20017A) plus Alexa Fluor^® 488 anti-mouse IgG (BioLegend, Clone Poly4053). Cells were stained for 45 min at 4°C. Flow cytometry was performed on an LSR II flow cytometer (BD Biosciences). Data were analyzed with FlowJo software (Tree Star Inc.).