Donkey anti rabbit igg brilliant violet 421

Ultra-thin sections (5 µm) were cut vertically and mounted on microscope slides. Nuclei were counterstained using Draq5 (BioLegend). Apoptotic cells were labeled using the TUNEL assay (In situ cell death detection kit, TMR red; Merck, Darmstadt, Germany) according to manufacturer’s specifications. Proliferating cells were labeled using an anti-Ki67 monoclonal antibody (primary antibody: rabbit anti-Ki67; Bethyl Laboratories, Montgomery, TX, USA) that was marked using a fluorescently labeled secondary antibody (donkey anti-rabbit IgG Brilliant Violet 421; BioLegend)). Microscopy slides were examined using a Keyence BZ-9000 fluorescence microscope (Keyence, Frankfurt, Germany). Using the software dynamic cell count (BZ-II Analyser, Keyence, Frankfurt, Germany), the ratio of TUNEL positive and negative cells (TUNEL⁺/Draq5⁺ vs. TUNEL^-/Draq5⁺) was determined [43 (link)].

Tissues were stained with the following antibodies: Primary/Conjugated antibodies: CD74 (Rabbit polyclonal, Abcam, #ab64772), CD8 (Mouse IgG2b, clone: 4B11, Thermo Scientific, #MA1-80231), ID3 (Mouse IgG1, clone: OTI8B3, abcam, ab236505), Ki67 Brilliant Violet 480 (BD Horizon, clone: B56, 566172), CD19 Alexa Fluor 647 (Biolegend, clone: A17136C, 396304), CD4 Alexa Fluor 700 (R&D systems, goat polyclonal, FAB8165N). Secondary antibodies: Donkey anti- Rabbit IgG Brilliant Violet 421 (Biolegend, clone: poly4064; 406410), Goat anti-Mouse IgG2b Alexa Fluor 488 (Life Technologies, A21141), Goat anti-Mouse IgG1 Alexa Fluor 594 (Life Technologies, A21125). Mouse IgG1 kappa isotype control clone B11/6, abcam, ab91353.