Protein detection of CD3, CD8, CD20, cytokeratin, and 4’,6-diamidino-2-phenylindole (DAPI) were simultaneously quantified on the same slide for every patient, as previously detailed by Brown et al., [
14]. Briefly, fresh cuts of whole tissue sections were deparaffinized and rehydrated before undergoing antigen retrieval using an EDTA buffer (pH = 8) for 20 minutes at 97 °C (PT module, Lab Vision, Thermo Fisher Scientific, Waltham, MA, USA). Slides were then incubated with dual endogenous peroxidase block (Dako, Glostrup, Denmark) for 10 minutes to block endogenous peroxidase activity and then incubated with 0.3 % bovine serum albumin in a 0.05 % Tween solution for 30 minutes to block nonspecific antigens. Fluorescent staining for pancytokeratin, CD3, CD8, and CD20 was performed using a sequential multiplexed protocol with different isotype-specific primary antibodies. Antibodies against these targets were used to detect epithelial tumor cells (cytokeratin, clone M3515, Dako), all T lymphocytes (CD3 IgG, clone E272, Novus Biologicals, Littleton, CO, USA), cytotoxic T lymphocytes (CD8 IgG1, clone C8/144B, Dako), and B lymphocytes (CD20, IgG2a, clone L26, Dako). All nuclei were then tagged with DAPI (Life Technologies, Carlsbad, CA, USA). Secondary antibodies conjugated to horseradish peroxidases (HRPs) and specific to each primary antibody isotype were used (anti-rabbit EnVision, Dako; anti-mouse IgG1, eBioscience, San Diego, CA, USA; anti-mouse IgG2a, Dako), while tyramide-bound fluorophores were added to bind to the HRPs (biotinylated tyramide, PerkinElmer, Waltham, MA, USA; streptavidin-Alexa750, Life Technologies; TSA™Plus fluorescein-tyramide, PerkinElmer; cyanine 5, PerkinElmer, respectively). A fluorophore-conjugated goat anti-rabbit secondary antibody was used against the cytokeratin antibody (Goat anti-Rabbit Alexa546, Life Technologies). Residual, unbound HRPs were blocked between incubations with a 0.15 % hydrogen peroxide benzoic hydrazide solution.
Slides were stained in three batches with a LabVision autostainer, in which samples from 10 to 11 patients were stained in each run. All biopsies from the same tumor were stained in the same batch to reduce experimental variability of expression of each target within patient samples. Morphologically normal human tonsil whole tissue sections were included in each batch as lymphocytic-positive control slides and to account for any variability in protein expression between batches. Additional file
1: Figure S1 shows small batch to batch variation for each marker.