The results indicated that DSBs triggering deletions in lymphoid leukemia are formed at a few defined sites by illegitimate action of the RAG protein complex, while DSBs in solid tumors are formed at unspecific sites by factors unidentified yet.
The islets were then washed (3 times) and fixed with 4% formaldehyde, permeabilized with 5% Triton X-100, and unspecific sites were blocked with 5% Normal Donkey Serum (Jackson Immunoresearch Laboratories Inc, West Grove, PA).
Islets freshly isolated or collected after incubation in KRB buffer as described above were fixed with 4% formaldehyde, permeabilized with 5% Triton X-100, and unspecific sites blocked with 5% Normal Donkey Serum (Jackson Immunoresearch Laboratories Inc, West Grove, PA, USA).
aOthers: retroperitoneum and unspecific sites.
bOthers: retroperitoneum and unspecific sites.
Unspecific sites were blocked by incubating cells in 1% of bovine serum albumin (BSA) at room temperature.
Blocking of unspecific sites was achieved by incubation with 10% donkey or goat serum (Sigma-Aldrich, St . Louis MO, USA) for 1 h at room temperature.
The increase in the incidence of GIST from esophagus/others resulted mostly from the elevated incidence of GIST located in retroperitoneum and unspecific sites (separate data not shown).
For immunohistochemistry, after blocking endogenous peroxidase activity with 3% hydrogen peroxide, the sections were incubated with blocking buffer to further block unspecific sites.
Plates were washed three times in PBS containing 0.05% (V/V) Tween-20 (PBS/Tween) and then incubated in 1% bovine serum albumin (BSA) in PBS for 1 h at RT to block unspecific sites.
