Dako autostainer plus

4% PFA-fixed paraffin-embedded tissues were sectioned at 5 μm and mounted onto Apex Superior Adhesive Slides (Leica, Wetzlar, Germany). Standard deparaffinization was done in xylene and serial alcohol immersion. Heat-induced epitope retrieval was performed in eBioscience IHC Antigen Retrieval Solution, pH 6.0 (Thermo Scientific) by pressure cooking at 125°C for 30 s and gradual cooling to 90°C over 40 min. Slides were stained using Dako Autostainer Plus (Dako Omnis; Agilent, Santa Clara, CA, United States) slides were rinsed with buffer and blocked with H₂O₂ block for 10-min, Dako envision kit Protein Block (Dako Omnis). Slides were incubated with S100A9 primary antibody (1:3000 dilution, NB110–89726; Novus Biologicals) for 1 h with a posterior 30-min Dako Rabbit Labeled Polymer secondary antibody (Dako Omnis), washed (×2) for 7-min with Dako DAB + Chromogen (Dako Omnis), and counterstaining with blue Mayer’s Hematoxylin (Sigma Aldrich). Slides were imaged and scanned using a NanoZoomer 2.0-HT. Images were analyzed by NDP.view2 Viewing software (Hamamatsu Photonics, Hamamatsu, Japan) and ImageJ.

Rabbit polyclonal antibody specific for PRR [ref. HPA003156; Sigma-Aldrich (Saint Louis, MO, USA) at 1/50 dilution] was used for the immunostaining of formalin-fixed and paraffin-embedded tumour tissues. The antibody’s specificity was tested previously [27 (link)] and the immunostaining process was performed following routine methods in an automatic immunostainer (Dako Autostainer Plus, Dako-Agilent, Santa Clara, California, USA). Briefly, antigen retrieval was carried out in a low-pH buffer (K8005, Dako, Santa Clara, California, USA) for 20 min at 95 °C. The samples were incubated with the primary antibody for 50 min at room temperature. Then, the primary antibody was washed and samples were incubated for 20 min with secondary anti-rabbit antibody (K8021, Dako). EnVision-Flex detection system together with an HRP-enzyme-labelled polymer (SM802, Dako) was employed. A positive reaction was visualized with diaminobenzydine (DAB) solution (DM827, Dako), followed by counterstaining with haematoxylin (K8008, Dako).
Slides were reviewed under light microscopy for staining evaluation. Two observers independently evaluated the slides and, in the event of discrepancies, samples were re-evaluated to arrive at a final conclusion. As previously reported [27 (link)], staining patterns were scored as negative, weak and intense cytoplasmic and membranous staining.

Immunohistochemistry was performed on 5-μm thick sections from formalin-fixed paraffin embedded tissue. Glass-mounted sections were de-parafinized in xylene and rehydrated in ethanol and dH₂O. Rabbit polyclonal antibodies used for DPR immunohistochemistry were as follows: poly-GA (Rb9880), poly-GP (Rb5823), poly-GR (Rb7810) (Leonard Petrucelli, Mayo Clinic, Jacksonville, FL [1 (link), 11 (link)]). A rabbit polyclonal antibody (ASYM24 07–414, MilliporeSigma, Burlington, MA) was used for immunohistochemistry for aDMA. Regions of interest were middle frontal gyrus (FCtx), motor cortex (MCtx) and hippocampus. Adjacent sections were immunostained for phospho-TDP-43 as above. All Immunoperoxidase sections were processed on a DAKO AutostainerPlus (Agilent/DAKO, Santa Clara, CA) with the DAKO EnVisionTM+ system-HRP with diaminobenzidine as the chromogen. Nonspecific antibody binding was blocked with normal goat serum (Sigma, St Louis, MO).
For double immunofluorescence staining, a rat monoclonal antibody was used to detect poly-GR (5H9, MilliporeSigma, Burlington, MA) and a rabbit polyclonal antibody was used to detect aDMA (MilliporeSigma, Burlington, MA). The fluorochromes were Alexa Fluor 568 and Alexa Fluor 488 conjugated to anti-rabbit or anti-rat IgG (Invitrogen/ThermoFisher, Waltham, MA).

Expression of the immune checkpoint molecules in CLL and RS lymph node samples was analyzed by IHC staining using antibodies specific for PD-L1 and PD1. Immune cell infiltration in CLL and RS lymph node samples was assessed by IHC staining using antibodies specific for CD3, CD8, FOXP3, and CD163. IHC staining was done on 4-μm FFPE sections on DAKO Autostainer Plus (Agilent, Santa Clara, CA) using standard protocol^{17 (link),20 (link)}. The antibodies used include anti-PD-L1 (clone SP263, Ventana Medical Systems, Inc., Tucson, AZ), anti-PD1 (clone NAT105, Abcam, Inc., Cambridge, MA), anti-CD3 (clone LN10, Leica Biosystems, Newcastle Upon Tyne, UK), anti-CD8 (clone C8/144B, Dako, Carpenteria, CA), anti-FOXP3 (clone 236A/E7, Abcam, Inc., Cambridge, MA), and anti-CD163 (clone10D6, Leica Biosystems, Newcastle Upon Tyne, UK). IHC images were taken using whole slide imaging technology with MoticEasyScan Pro (Motic digital pathology, San Francisco, CA), and saved in tagged image file format. Percentage of expression for each individual antigen was calculated by dividing number of cells with positive staining by number of total cells in the image using the Image-Pro premier 3D 9.1.4 software (Media Cybernetics, Silver Spring, MD).