Biotin

Iterative rounds of immunostaining started with the first round of immunohistochemistry, as described above. After the microscope slide scanning, the coverslip was removed by soaking the slide in water, typically overnight on a shaker, until the coverslip fell off. Next, the chromogen was removed as described above. Heat-induced antigen retrieval was then completed as described previously, followed by a series of blocking steps. First, the slides were incubated for 30 min in 0.4% Triton-X 100. Then endogenous peroxidases were quenched with 0.3% H₂O₂ in methanol for 30 min. The samples were then incubated for 60 min in blocking buffer (10% normal goat serum, with 0.2% Triton-X 100, in tris buffer saline) at room temperature. Residual avidin and biotin was blocked by incubating in avidin (Vector Laboratories) diluted in blocking buffer for 1 h, washed three times in blocking buffer, and then incubated in biotin (Vector Laboratories) diluted in blocking buffer for 1 h. If the same species primary antibody was used, the samples were incubated in fragment antigen-binding region (FAB) (Jackson Immuno Research labs) overnight at 4 °C. For the remaining steps, we used the immunohistochemistry protocol described above. For each round of staining a no primary antibody control and positive control samples were included.