Ab27988

The tumor tissues fixed in 4% paraformaldehyde (at room temperature for 24 h) were embedded in paraffin and then sectioned into 5-µm-thick slices. Immunohistochemistry was performed using an Instant SABC-POD kit (cat. no. SA1020, Boster Biological Technology) according to the manufacturer's instructions. Briefly, after dewaxing, rehydration and antigen retrieval, the tumor sections were blocked and were then separately incubated with primary antibodies against pan-cytokeratin (AE1/AE3; cat. no. ab27988, Abcam, dilution: 1:200) and CPT1A (cat. no. ab128568, Abcam, dilution: 1:200) overnight at 4°C. After washing, the sections were incubated with biotin-conjugated secondary antibody (cat. no. SA1020, Boster Biological Technology) at 37°C for 30 min. After washing, the sections were incubated with SAB at 37°C for 30 min and stained with DAB (cat. no. AR1027, Boster Biological Technology) at room temperature for 5 min. Following DAB visualization, the sections were immersed into water and then re-stained using hematoxylin at room temperature for 3 min. The AE1/AE3-positive cells or the CPT1A-positive cells were finally observed under a light microscope (Nikon Corporation).

Balb/c mice were purchased from Harlan (UK) and were housed and kept in specific pathogen-free sterile conditions, at the Transgenic Mouse Facility (TMF) of the Cyprus Institute of Neurology and Genetics in Nicosia, Cyprus, which is licensed by the Cyprus Veterinary Services (C.EXP.101). All the bedding and water for the mice were sterilized by autoclaving. The experiments were performed under the animal project license (CY/EXP/PR.L6/2011) provided to A.I.C., issued and approved by the Cyprus Veterinary services, which is the Cyprus national authority for monitoring animal research for all academic institutions according to the regulations contained in the Cyprus Law N.55 (I)/2013 and the EU Directive 2010/63/EU. Two groups of fourteen (14 (link)) female (6-8-week-old) Balb/c mice (15-20 g weight) were inoculated by injecting 1–5×10⁶ either normal HC11 or massive clone retrotransposition-positive cells per group into the fat pads near the posterior mammary gland. Developed tumors along with surrounding tissues were removed, immediately fixed in 10% neutral-buffered formalin for 24 h and embedded in paraffin using standard procedures. Paraffin-embedded tumors were cut in 5-µm-thick sections, stained either with hematoxylin-eosin alone or analyzed by immunohistochemistry using a 1:300 diluted anti-pan cytokeratin antibody [AE1/AE3] (ab27988, Abcam) and hematoxylin staining.

A 5-μm tissue section adjacent to the section used for ISH was probed for rabbit polyclonal cytokeratin 5 (KRT5, clone Poly19055, BioLegend, San Diego, CA) and mouse monoclonal pan-cytokeratin AE1/AE3 (ab27988, Abcam, Cambridge, UK) antibodies diluted to 1:200. Antigens were retrieved using sodium citrate buffer, pH 6, 100°C for 5 minutes at 5 psi. Alexafluor 555– and 488–labeled secondaries (Invitrogen, Carlsbad, CA) were used at 1:200, followed by DAPI nuclear counterstain. Slides were imaged on the Vectra Automated Multispectral Imaging System (PerkinElmer, Waltham, MA) at the Research Histology and Tissue Imaging Core at UIC. The other adjacent section was hematoxylin and eosin (H&E) stained and scanned with Aperio AT2 (Leica, Wetzlar, Germany) at the Research Histology and Tissue Imaging Core.