Retrievit 6

Tissues were fixed in 4% paraformaldehyde at 4°C, processed according to standard protocols, and embedded in paraffin. Antigen retrieval was performed using heat-mediated microwave methods and an antigen unmasking solution (Vector Laboratories, H-3300) for all antibodies except rat-anti-CD45, for which Retrievit 6 (BioGenex, BS-1006-00) was used. All sections were blocked in 10% FBS in PBST and primary antibodies were incubated overnight at 4°C. Secondary antibodies, from Jackson Immunoresearch, were used at 1:250 and incubated at room temperature for 2 hours for IF and 30 min for IHC. For IF, slides were stained with IHC-Tek Dapi counterstain solution (IHC World, IW-1404) and mounted in fluorescence mounting medium (Dako, S3023). For IHC, Vectastain Elite ABC kit (Vector Laboratories, PK-6100) and DAB Peroxidase (HRP) Substrate kit (Vector Laboratories, SK-4100) were used. Primary antibodies used in this study are described in Supplemental Table 1.

Antigen retrieval for Aldh1 immunohistochemistry was accomplished using Retrievit 6 (BS-1006-00, BioGenex). For Cytokeratin 19 immunolabeling, antigen retrieval was done by digesting sections with 250 μg/mL proteinase K in 2.5 mmol/L CaCl₂ and 10mmol/L Tris-HCl (pH 7.5) for 6 minutes at room temperature. For NF-kB immunohistochemistry, antigen retrieval was accomplished using R-Buffer A (62706-10, Electron Microscopy Sciences). Antigen retrieval for CD45 was accomplished using a citrate based antigen retrieval buffer and a heat-mediated microwave method. Immunohistochemistry staining was visualized on an Olympus BX40 light microscope.