Dylight 488 affinipure goat anti mouse igg

After deparaffinization, rehydration and washing of the sections, microwave antigen retrieval was carried out in Tris‐EDTA (pH 9.0). Sections were first incubated with blocking serum at room temperature for 30 minutes and with the mouse anti‐IDO1 mAb (4.16H1 Ab, 1:1000; a kind gift from Benoit J. Van den Eynde), combined with a rabbit anti‐CD34 mAb (EP373Y Ab 1:100; #ab81289, Abcam), a rabbit anti‐LAMP3 mAb (104G4 Ab, 1:50; #DDX0190A546‐100, Novus Biologicals), or a rabbit anti‐CD68 polyclonal Ab (1:100; #25747‐1‐AP, ProteinTech Group) at 4°C overnight. The second incubation was undertaken using DyLight 488 AffiniPure goat anti‐mouse IgG (1:100; #E032210, EarthOx, Millbrae, CA, USA) and DyLight 594 AffiniPure goat anti‐rabbit IgG (1:100; E032420, EarthOx). Following counterstaining with DAPI after serially rinsing with 1× PBST, images were acquired in a microscopic field at 600×. The observation and images acquisition of these sections were carried out using a confocal laser scanning microscope (Olympus Fluoview FV1000; Olympus, Tokyo, Japan).

Formalin-fixed, paraffin-embedded tissue samples were cut into 5-µm sections and mounted on glass slides. After the sections were deparaffinized, rehydrated, and washed, microwave antigen retrieval was performed in Tris-EDTA (pH 9.0). Sections were first incubated with blocking serum at room temperature for 30 min and with mouse anti-IL-6 monoclonal antibody (1:1,000, ab9324; Abcam) combined with rabbit anti-CD11b monoclonal antibody (1:200, ab52478; Abcam) at 4 °C overnight. The second incubation was done using DyLight 488 AffiniPure goat anti-mouse IgG (1:100, E032210; EarthOx) and DyLight 594 AffiniPure goat anti-rabbit IgG (1:100, E032420; EarthOx) after counterstaining with DAPI and serial rinsing with 1× PBST.