Fig. 2

Blocking step optimization to limit antibody cross-reactivity. (A) Experimental workflow for antibody cross-reactivity test: (1) Indirect IHC is completed using an ethanol-soluble chromogen. (2) The digital slide is generated using a slide-scanning microscope. (3) The chromogen is removed using the chemical de-staining method. (4) The slide is then visually inspected to confirm the de-staining was ~ 100% efficient. (5) Additional blocking steps are added to limit cross-reactivity. (6) The tissue is stained following step 1, omitting the primary antibody. (7) A digital slide is created, and (8) digital pathological tools are used to quantify the percentage of cross-reactivity by co-registration and direct comparison of the image from step 2 to the image in step 8. (B) Avidin and biotin (A&B) blocking conditions were tested (Table 2). The photomicrographs show a comparison of the GFAP staining between the best A&B block condition versus the omission of the A&B blocking step. Digital neuropathological quantification of the area fraction of GFAP⁺ staining show that approximately 35% of the staining remains if the A&B blocking step is omitted. (C) The effect of varying the FAB blocking conditions (Table 2) were tested while holding the best A&B blocking condition from B constant. A high degree (46%) of GFAP⁺ staining was seen in samples lacking the FAB blocking step following re-staining whit the secondary antibody alone. By area fraction digital quantification, the degree of re-development of the GFAP staining was further blocked beyond the optimized A&B conditions using a higher concentration of FAB a incubating the samples in a greater volume of the blocking solution