Dako pt link pre treatment module

PD‐L1 IHC was carried out on 4‐μm sections of FFPE tumour tissue samples using Dako PD‐L1 IHC 28‐8 PharmaDx (Agilent). The test was performed using the EnVision FLEX visualization system on the Dako Autostainer Link 48 and Dako PT Link Pretreatment Module (Agilent). A minimum of 100 viable tumour cells must be present for evaluation. PD‐L1 expression was evaluated only in tumour cells. Scoring was determined according to the tumour proportion score (TPS), which is defined as the percentage of positive viable tumour cells among all viable tumour cells evaluated. A tumour cell was defined as positive for PD‐L1 staining whenever any partial or complete membranous staining was detected. The percentage of PD‐L1‐positive tumour cells was assessed as previously described [20 (link)]. Slides were assessed independently by two pathologists.

Lung tissue sections were fixed as reported above. Lung samples underwent antigen retrieval (pH 9 buffer) using a Dako PT Link pre-treatment module (Agilent). Samples were washed and blocked for 10 min (Dako protein block; Agilent, Santa Clara, CA) before being treated with primary antibodies overnight. Mouse anti-COL1A1, ARG1, and rabbit anti-FDPS, CD206 (Abcam, Cambridge, United Kingdom) antibodies were used. Alexa Fluor 488-conjugated goat/anti-mouse and Alexa Fluor 647 goat/anti-rabbit (Invitrogen, Waltham, CA, United States) were used as secondary antibodies. Glass cover slips were placed onto slides and mounted with DAPI-containing fluoroshield (Abcam). Microscopy was performed on a Widefield Epifluorescence Ti2 microscope equipped with a Nikon DS-Qi2 camera and fluorescence was quantified using ImageJ software.

Fixated lung tissue sections underwent antigen retrieval (pH 9 buffer) using a Dako PT Link pre-treatment module (Agilent, Santa Clara, CA). Samples were washed with PBS and blocked with Dako protein block (Agilent) for 10 min, followed by incubation with primary antibodies overnight (mouse anti-mouse arginase-1 (ab239731), mouse anti-mouse MUC5ac (MA5-12178)). Samples were incubated with secondary antibodies, Alexa Fluor 594 goat anti mouse (Abcam, Cambridge, United Kingdom). Glass cover slips were mounted with DAPI- containing fluoroshield (Abcam). Images were visualized using a Nikon Confocal Microscope (Nikon, Tokyo, Japan) and fluorescence was quantified using ImageJ software (https://imagej.nih.gov).