Immunohistochemistry was performed on formalin fixed, paraffin-embedded tissue sections. Tissue microarrays were conducted using the primary tumour specimens[18 (link)], and a human kidney tissue specimen was included in the multi-tissue block as a positive control for TLR2 staining[19 (link)]. Macrophages served as positive control for TLR4 staining. Samples representing the normal mucosa and lymph node metastases were stained and analysed separately.
For antigen retrieval, sections were treated at a high temperature in Tris-EDTA buffer for 15 min. Immunostaining was performed with Dako Autostainer (Dako Copenhagen, Denmark) using mouse monoclonal antibodies (Abnova MAB0066 Clone 1030A5.138 for TLR2 and Abnova H00007099-M02 Clone 3B6 for TLR4; Abnova, Taoyuan City, Taiwan), at dilutions of 1:50 and 1:1000, respectively. For detection, we used Dako Envision kit (Dako) and diaminobenzidine (Dako basic DAB-kit) as a chromogen. For negative controls, we omitted the primary antibody and replaced the primary antibody with a mouse primary antibody isotype control.
For antigen retrieval, sections were treated at a high temperature in Tris-EDTA buffer for 15 min. Immunostaining was performed with Dako Autostainer (Dako Copenhagen, Denmark) using mouse monoclonal antibodies (Abnova MAB0066 Clone 1030A5.138 for TLR2 and Abnova H00007099-M02 Clone 3B6 for TLR4; Abnova, Taoyuan City, Taiwan), at dilutions of 1:50 and 1:1000, respectively. For detection, we used Dako Envision kit (Dako) and diaminobenzidine (Dako basic DAB-kit) as a chromogen. For negative controls, we omitted the primary antibody and replaced the primary antibody with a mouse primary antibody isotype control.
