Goat anti mouse igg2b alexa 647

4-µm serial sections from paraffin-embedded tissue were cut and used for immunostaining. Immunohistochemistry (IHC) of BAP1 was performed on 74 tumors, as described previously [24 (link)]. Tumors were scored by two independent investigators as BAP1-positive or -negative based on nuclear staining. Immunofluorescence (IF) staining for T cells and macrophages was performed on 43 tumors as described previously [29 (link), 30 (link)]. T cell types were detected by primary antibodies: anti-CD3 (ab828, rabbit polyclonal; Abcam, Cambridge, MA, United States of America) and anti-CD8 (4B11, mouse monoclonal IgG2b; Novocastra, Valkenswaard, The Netherlands). To visualize the T cells, the following secondary antibodies were used: goat-anti-rabbit IgG Alexa 546 and goat-anti-mouse IgG2b Alexa 647 (Molecular Probes, Invitrogen, Breda, The Netherlands). Counts of intratumoral CD3⁺ and CD8⁺ T cells were represented as the number of cells per square millimeter. For IF staining of CD68⁺ macrophages, we used the primary mouse anti-human macrophage CD68 antibody (clone 514H12; ab49777; Abcam, Cambridge, United Kingdom), and as secondary antibody AlexaFluor IgG2a (488) goat-anti-mouse. The amount of CD68⁺ expression was determined in pixels per square millimeter.

Characterization of lymphocytic infiltration was carried out with triple immunofluorescent staining in 41 OPSCC as described previously [10 (link)] using anti-CD8 (mouse IgG2b, clone 4B11; Novocastra, 1:400), anti-Tbet (rabbit polyclonal, clone H210; Santa Cruz 1:400) and anti-Foxp3 (mouse IgG1, clone 236A/E7; Abcam, 1:200), goat-anti-mouse IgG2b Alexa 647, goat-anti-rabbit Alexa 546 and goat-anti-mouse IgG1 Alexa 488 (all from Molecular probes; 1:200). Based on the morphology of cancer cell nests and autofluorescence of keratinocytes the immune cells per mm² were manually counted as intraepithelial or stromal using the LSM 5 Image Examiner software (average of five images at a 250× magnification).

OCs were grown from RA SFMCs on sterile glass slides in 24-well cell culture plates and stained for confocal microscopy as previously described [35 (link)]. Briefly, cells were fixed with 4 % paraformaldehyde for 10 minutes at RT. Non-specific binding was blocked by incubating in PBS with 0.5 % BSA and 5 % goat serum for 30 minutes at RT. Cells were stained with either anti-IL-20R1 IgG1 (173714; R&D Systems) or anti-IL-22R1 IgG1 (305405; R&D Systems) in combination with goat anti-mouse IgG1 Alexa 488 (Invitrogen). Cells were co-stained with anti-TRAP IgG2b in combination with goat anti-mouse IgG2b Alexa 647 (Invitrogen). Isotypes served as negative controls. Glass slides were placed in Prolong Gold Antifade Mountant with DAPI (Life Technologies) and allowed to dry overnight. All micrographs were collected using a Zeiss LSM-710 confocal microscope.