The mouse anti-human GARP (hGARP) antibody (ALX-804-867-C100, Enzo Life Sciences) was first verified by Western blot using hGARP-transfected HEK-293 cells and by IHC with hGARP-transfected 70Z/3 cells. Both analyses demonstrated specificity of the antibody and dilutions used from 1:250 (colon cancer) to 1:60 (all other cancers).
TMA slides were processed and antigen retrieved as described previously (33 (link)). For mouse IHC, tissue was either placed into OCT media for fresh frozen sections or fixed in 4% paraformaldehyde overnight. Fixed tissue was incubated in 70% ethanol overnight prior to paraffin embedding, and then cut for hematoxylin and eosin (H&E) staining. For p-Smad-2/3 on fresh frozen tumor sections, 5 μm sections were fixed with 4% paraformaldehyde followed by incubation with 3% H2O2. To minimize nonspecific staining, sections were incubated with the appropriate animal serum for 20 min at room temperature, followed by incubation with primary anti-p-Smad-2/3 antibody (EP823Y; Abcam) overnight at 4°C. Staining with secondary antibodies (Vectastain ABC Kit) was then performed before development using DAB substrate (Vector Labs SK-4100). The staining intensity of GARP and pSmad-2/3 was graded as follows with the sample identity blinded (0: negative; 1: faint; 2: moderate; 3: strong but less intense than 4; and 4: intense).
TMA slides were processed and antigen retrieved as described previously (33 (link)). For mouse IHC, tissue was either placed into OCT media for fresh frozen sections or fixed in 4% paraformaldehyde overnight. Fixed tissue was incubated in 70% ethanol overnight prior to paraffin embedding, and then cut for hematoxylin and eosin (H&E) staining. For p-Smad-2/3 on fresh frozen tumor sections, 5 μm sections were fixed with 4% paraformaldehyde followed by incubation with 3% H2O2. To minimize nonspecific staining, sections were incubated with the appropriate animal serum for 20 min at room temperature, followed by incubation with primary anti-p-Smad-2/3 antibody (EP823Y; Abcam) overnight at 4°C. Staining with secondary antibodies (Vectastain ABC Kit) was then performed before development using DAB substrate (Vector Labs SK-4100). The staining intensity of GARP and pSmad-2/3 was graded as follows with the sample identity blinded (0: negative; 1: faint; 2: moderate; 3: strong but less intense than 4; and 4: intense).
