Streptavidin biotin complex

Tissue sections (4μm thick) were immersed in citrate buffer solution for antigen retrieval and boiled in microwave for 10 min and washed in buffer solution. They were incubated with primary antibody including anti-CD8 (C8/144B, DAKO), anti-CD45RO (UCHL1, DAKO) and the anti-PDL1 mouse IgG1 (clone 5H1; Thompson) antibodies for 1 hr at room temperature and then washed in buffer solution. After 1 hr of incubation in the secondary antibody, the sections were incubated with streptavidin-biotin-complex (DAKO). Appropriate positive and negative controls were used. Stained tumor samples were reviewed and enumerated by a GI pathologist. For estimation of positive reaction, only the strongly stained nuclei were counted as a percentage of all tumor nuclei. Less than 1% were estimated as negative. Samples with greater than 1% of cells exhibiting strong nuclear staining were considered positive.

Lung tissues fixed in formaldehyde were dehydrated by a series of ethanol solutions with increasing concentrations and then embedded in paraffin. Sections of 5 μm thickness were prepared and dewaxed. After hydration, tissues were stained with hematoxylin and eosin (H&E) or periodic acid-Schiff (PAS). The observation of airway inflammation and goblet cell hyperplasia and mucus production was conducted as previously described [11 (link)]. In brief, peribronchial inflammation in airways was evaluated with an 8-point semiquantitative scoring system, while goblet cell hyperplasia was determined with Pierre Camateros' method by counting PAS-positive cells and standardized by dividing the perimeter of the basement membrane.
Immunohistochemistry for p-MTOR (Cell Signaling, Danvers, MA, USA) was performed on the hydrated sections. After antigen retrieval with citrate buffer (pH 6.0) by heating the slides for 15 min in a microwave oven, normal goat serum was applied for 30 min and then sections were incubated overnight at 4°C with the primary antibodies. A three-step technique (labelled streptavidin-biotin complex, Dako, Glostrup, Denmark) was used for visualization, and diaminobenzidine (DAB) used as a chromogen. Finally, the sections were counterstained with hematoxylin.