Anti mouse igg alexafluor 647

GCs and GC-resident T_FH cells were detected using immunofluorescence staining. Briefly, 6-μm sections were cut and adhered to silanized slides and stained with the following antibodies: mouse anti-human CD4 (1F6, Leica Microsystems Inc., Buffalo Grove, IL), goat anti-human PD-1 (AF1086, R&D Systems, Minneapolis, MN), or goat anti-human CD20 (MS4A1, Origene, Rockville, MD) and rabbit anti-human Ki67 (SP6, ThermoFisher Scientific). Sections on slides were pretreated in 1 mM EDTA (pH 8.0) in a Presto pressure cooker (National Presto Industries, Eau Claire, WI) at 121°C for 35 seconds to unmask antigens. Sections were blocked with normal horse serum, and incubated with primary antibodies overnight at 4 °C. After washing in PBS, the slides were incubated at room temperature for 2 h with donkey AlexaFluor 647 anti-mouse IgG, AlexaFluor 488 donkey anti-goat IgG and AlexaFluor 594 donkey anti-rabbit IgG and counterstained with DAPI (4',6-diamidino-2-phenylindole), all from Life Technologies (Carlsbad, CA). After washing in PBS, the slides were coverslipped and examined using a Nikon A1 confocal microscope system (Nikon, Melville, NY). GCs were enumerated and size quantified using ScanScope slide scanner and ImageScope software (Aperio-Leica Biosystems, Buffalo Grove, IL).

For interphase nuclei, cells were seeded onto glass coverslips 24 h before the experiment as described^{33 (link)}. Cells were washed in PBS, fixed in 4% formaldehyde in PBS for 10 min and permeabilized in 0.1% triton in PBS for 10 min. For metaphase spreads, cells were treated with 20 ng/ml colcemid (Gibco #15212–012) for 1 h, collected and incubated in hypotonic solution (27 mM KCl, 6.5 mM tri-sodium citrate) for 5 min. The cell suspension was cytocentrifuged, fixed in 4% formaldehyde in PBS for 10 min and permeabilized in KCM buffer (120 mM KCl, 20 mM NaCl, 10 mM Tris pH 7.5, 0.1% Triton) for 10 min.
In both settings, samples were incubated in blocking buffer (20 mM Tris pH 7.5, 2% BSA, 0.2% fish gelatin, 150 mM NaCl, 0.1% triton, 0.1% sodium azide, 100 μg/ml RNaseA) for 1 h at 37 °C. Cells were incubated with the primary antibody (γH2AX) for 2 h, washed in PBS and incubated with secondary antibody for 1 h at room temperature. Secondary antibodies used were AlexaFluor 568 anti-IgG mouse (ThermoFisher #A-11004) or AlexaFluor 647 anti-IgG mouse (ThermoFisher #A-21235). Samples were fixed in 4% formaldehyde in PBS before FISH.