For light microscopy, 4 μm thick sections from formalin-fixed paraffin-embedded tissue were automatically stained with hematoxylin and eosin in a Sakura
Tissue-Tek Prisma instrument (Sakura Finetek, Torrance, California, USA). The immunostainings were done on a
Dako Autostainer instrument (Dako, Agilent Technologies, Santa Clara, California, USA, model Link 48), and the incubation time for the primary antibodies was 20 min. CD3 was immunostained by clone SP7 (a monoclonal rabbit antibody, diluted 1:150; Thermo Scientific, Waltham, Massachusetts, USA; cat. no. RM-9107), and CD20 was immunostained by clone L26 (a mouse IgG2a antibody, diluted 1:600; Dako, cat. no. M0755). The secondary detection was performed with Dako EnVision
TM Flex (Dako, cat. no. K8000) for 20 min, followed by diaminobenzidine (DAB) staining for 10 min. The slides were thereafter treated with CuSO4 for 5 min before contrastaining with hematoxylin. Samples were examined with a Nikon Eclipse model N
i-U microscope (Nikon, Tokyo, Japan) equipped with Nikon Plan-Fluor objective lenses (2×, 20×, and 40×) and images were taken with an Infinity 2 digital camera (Lumenera Corporation, Nepean, Ontario, Canada).
Joly-Battaglini A., Hammarström C., Stankovic B., Aamodt H., Stjärne J., Brustugun O.T., Helland Å., Øynebråten I, & Corthay A. (2016). Rituximab efficiently depletes B cells in lung tumors and normal lung tissue. F1000Research, 5, 38.