Bloxall endogenous blocking solution

Paraffin-embedded tissue samples were sectioned at
a thickness of
3–4 μm on charged microscope slides. Samples were deparaffinized
using CitriSolv (Decon Laboratories, Inc., King of Prussia, PA) and
rehydrated sequentially in graded alcohols. Antigen retrieval was
performed by incubating slides in 10 μg/mL proteinase K (MilliporeSigma)
in PK buffer (0.6 M Tris (pH 7.5)/0.1% CaCl₂) for 10 min
at RT. Blocking of endogenous peroxidase and alkaline phosphatase
was performed by incubating slides in Rodent Block M (BioCare Medical,
Pacheco, CA) followed by incubation with BLOXALL Endogenous Blocking
Solution (Vector Laboratories, Burlingame, CA). Slides were washed
in TBS buffer and incubated with primary rabbit Salmonella O Antiserum
(Group B Factors 1, 4, 5, 12, #BD 229481, Becton, Dickinson and Company)
for 1 h at 1:5000 dilution. Slides were then incubated in AP-polymer
(Rabbit on Rodent AP-Polymer, BioCare Medical) followed by Vina Green
Chromogen (BioCare Medical, Pacheco, CA) treatment. Tissues were finally
counterstained with hematoxylin (BioCare Medical) before mounting
with EcoMount (BioCare Medical). Slides were cured at 60–70
°C for 15 min.

Tumors were paraffin-embedded and 4 μm sections were used for immunohistochemistry. The sections were deparaffinized and treated with BLOXALL endogenous blocking solution (Vector Laboratories, CA, USA) to inhibit endogenous peroxidase activity. The epitope retrieval was conducted with Retrieve-All Antigen 1 (pH 8), followed by blocking with PBS supplemented with 2% normal horse serum and 3% BSA. Sections were incubated with individual primary antibodies at 4 °C overnight and with the appropriate secondary antibody at room temperature for 1 h. Peroxidase activity was revealed using 3,3-diaminobenzidine (Vector Laboratories), and sections were counterstained with hematoxylin (Vector Laboratories). After dehydrogenation with alcohol and xylene, sections were viewed and photographed with an Olympus BX51 microscope (Olympus, Tokyo, Japan) and Mosaic2.1 software. The following antibodies were used: Ki-67 (1:100, Abcam, Cambridge, UK), cleaved caspase-3 (1:100, Cell Signaling Technology, MA, USA), and KIT (1:200, Abcam). Ki-67, cleaved caspase-3, and KIT. Positive staining in xenografted tumors were quantified using ImageJ software (NIH). Three HPF (high-power fields, at ×400 magnification) were used for each tumor [42 (link)].