Id1 rabmab

Immunohistochemistry for ID1, ID2, ID3, and E-cadherin was performed on the TMAs, using a two-step protocol (EnVision™ Detection Systems, Dako, Glostrup, Denmark). The TMAs were dewaxed with xylene, gradually hydrated with gradient ethanol, and washed with phosphate-buffered saline (PBS). Antigens were retrieved by boiling the TMAs in citrate buffer (pH 6.0) at 121°C for 15 minutes. Endogenous peroxidase was blocked by incubating the TMAs in 3% H₂O₂ solution. Then, the slides were washed in PBS for 10 minutes, blocked with 10% normal goat serum (CW0130; CWBIO, Beijing, People’s Republic of China) for 30 minutes at 37°C, and incubated with Anti-ID1 (ID1 RabMab^®; Epitomics, Inc., Burlingame, CA, USA), anti-ID2 (ID2 [D39E8] Rabbit mAb; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-ID3 (Abgent, San Diego, CA, USA), anti-E-cadherin (BD Biosciences Pharmingen, San Diego, CA, USA) at 4°C overnight. The antibody dilution was 1:200 for the ID antibodies and 1:400 for E-cadherin antibody. The immunoreactive products were visualized by the catalysis of 3, 3-diaminobenzidine by horseradish peroxidase (EnVision™ Detection Systems), following extensive washings. The TMAs were then counterstained with Gill hematoxylin (BA-5021, Zhuhai, People’s Republic of China) and dehydrated in ascending grades of ethanol, and finally, cleared in xylene and mounted under a coverslip.

Whole cell lysates were generated using Pierce T-PER^® Tissue Protein Extraction Reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA) with complete EDTA (ethylene-diaminetetraacetic acid)-free Protease Inhibitor Cocktail Tablets (F Hoffman-La Roche Ltd, Basel, Switzerland). Immunoblotting was done, essentially as described.30 (link) The primary antibodies used were as follows: Anti-ID1 (ID1 RabMab^®; Epitomics, Inc., Burlingame, CA, USA), anti-ID2 (ID2 [D39E8] Rabbit mAb; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-ID3 (Abgent, San Diego, CA, USA), anti-E-cadherin (BD, Biosciences Pharmingen, San Diego, CA, USA), and anti–glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (GAPDH [6C5] mAb; Bioworld Technology, Inc., Minneapolis, MN, USA). The quality of loading and transferring was assessed by immunostaining with the GAPDH antibody. The dilutions for the antibodies were as follows: 1:1000 for ID1, 1:2000 for ID2, 1:1000 for ID3, 1:1000 for E-cadherin, and 1:2000 for GAPDH.