Immunohistochemistry
9167S
SECONDARY
Five-µm-thick frozen sections were cut from lung pieces and fixed with acetone for 5 min. Thereafter, the endogenic peroxidase was blocked using Bloxall Blocking solution (Vector laboratories) for 15 min at room temperature RT. The first-stage antibodies were against alpha and beta chains (IFNAR1 and IFNAR2) of the IFN α/β receptor (St John's Laboratory STJ112765 and LS-Bio LS-B13369, respectively). The anti-alpha chain antibody was used 1:2000 and 1:5000, anti-beta chain 1:1000, anti-STAT1 (1:400, 9175S), anti-pSTAT1 (1:100, 9167S), anti-STAT2 (1:200, 72604S), and anti-pSTAT2 (1:50, 88410S) all from Cell Signaling. Rabbit Ig (1 µg/ml) was used as a negative control. The secondary step was performed with Vectastain Elite ABC rabbit IgG kit (Vector laboratories) and diaminobenzidine was used as a chromogen.Two different concentrations of the antibody against the alpha chain of the IFN α/β receptor were used to better recognize the differences between the samples. The staining intensity was blindly scored from 0 to 3 and the mean intensity obtained by these two concentrations was used as the score for the alpha chain; mean of this and that obtained for the beta chain intensity (0 to 3) is shown for IFNAR. The intensity of the STAT stainings was semi-quantitatively scored from 0 to 3 both at the whole cell level and separately in nuclei of positive cell types. CD73 was detected with a monoclonal anti-CD73 antibody (4G4) 10 µg/ml followed by Alexa Fluor488 conjugated goat anti-mouse IgG (Invitrogen). A negative class matched monoclonal antibody was used as a control. Number of positive vessels were counted and the staining intensity was scored from 0 to 4. On average of 20 fields/sample at 200× magnification were evaluated. All samples were treated in the similar way and analyzed blindly without knowledge of the patient identity.