Publication protocol
Normal mouse tissues were fixed in formalin for a minimum of three days, and then processed and embedded in paraffin. Four-micron sections were cut, baked at 60C for at least 30 min and cooled. Sections were deparaffinized in three changes of xylene for 5 min each and then rehydrated to distilled water using graded alcohols. If required, HIER was used at this time by steaming the slides for 20 min, cooling them for 20 min, and then transferring the slides into wash buffer for 5 min. The antigen retrieval buffers used were citrate buffer at pH 6.0, Tris-EDTA buffer at pH 9.0, or Trilogy. The slides were transferred to TBS-Tween (TBS-T) wash buffer (final concentration 0.05 M Tris, 0.15 M NaCl, 0.05% Tween-20, pH 7.6 ± 0.1) for 5 min, and then loaded onto the Dako Autostainer Plus. Our antibodies were diluted in TBS containing 1% BSA at pH 7.5 ± 0.1. Endogenous peroxidase was blocked with a 3% hydrogen peroxide solution for 8 min (#33502-644, VWR International; Radnor, PA) (see Table 1). If needed, endogenous biotin was blocked with Avidin/Biotin System (#AB972M, Biocare Medical; Concord, CA) for 10 min each. When we used the biotinylated goat anti-mouse secondary antibody, we used 15% normal goat serum (Jackson ImmunoResearch Catalog # 005-000-121) in TBS with 1% BSA for 10 min as the protein block. When the secondary antibody was not biotinylated (either derived from goat or rabbit), we used TCT buffer (0.05 M Tris, 0.15 M NaCl, 0.25% Casein and 0.1% Tween-20) for 10 min as the protein block. To develop the signal, we used two sequential applications of 3, 3’-diaminobenzidine using the DAB Plus (#K3468, Dako) for 4 min each, followed by a 2 min incubation of hematoxylin as the counterstain (Automation Hematoxylin #S3301, Dako). After staining, the slides were dehydrated in graded alcohols, cleared in xylene, and coverslipped.
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