Envision flex mouse linker

Dewaxing, rehydration, and antigen retrieval was performed using a combined 3-in-1 Dako PT link module (Dako), and immunohistochemistry was performed manually by LH. Endogenous peroxidase activity was inhibited using EnVision FLEX Peroxidase-Blocking Reagent (Agilent Dako) for 60 minutes. Primary antibodies were incubated for one hour at room temperature: Ki67 at 1:400 (mouse monoclonal anti-Ki67, clone MIB-1, Cat no. M7240, Agilent Dako); γH2AX at 1:1000 (mouse monoclonal anti-gamma H2AX (phospho-S139) antibody, clone 9F3, Cat no. Ab26350, Abcam); and p21 at 1:100 (mouse monoclonal anti-p21, clone SX118, Cat no. M7202, Agilent Dako). For samples incubated with p21 antibody, an additional step involving 15 minutes incubation with EnVision FLEX mouse linker (Agilent Dako) was found to be optimal. An HRP-conjugated secondary antibody was applied for 30 minutes at room temperature (Dako EnVision FLEX/HRP, Agilent Dako), followed by incubation for five minutes with 3,3’-diaminobenzidine chromogen (Agilent Dako) and counterstain with Mayer’s haematoxylin for five minutes. Sections of canine skin were used as a positive control for each antibody (for example, as in (Kortum et al. 2018 (link))), and negative control slides were prepared using commercial species-matched immunoglobulins (Agilent Dako).

First, 4-μm-thick sections were mounted on poly-L-lysine coated slides, deparaffinized in xylene, and rehydrated using graded alcohols. Antigen retrieval was accomplished with a 10 mM citrate buffer (pH 6.0) at 95–98 °C for 20 min. Endogenous peroxidase was neutralized using EnVision FLEX peroxidase-blocking reagent (Dako, Denmark) for 10 min. Tween-phosphate-buffered saline (PBS) was used as the washing solution. Tissue sections were blocked with 3% bovine serum albumin and the mouse-on-mouse staining protocol (Abcam) was used for mouse samples with eNOS mouse-produced antibody. Anti-eNOS (6H2) (1:200, Cell Signaling, Danvers, MA, USA, ref 02/2016, lot 2, RRID AB_10850618), anti-iNOS (1:100, Invitrogen, lot RJ2276825, RRID AB_2537941), anti-Bmi1 (1:400, Abcam, lot GR301384-1, RRID AB_2065390), and β-catenin (BD, East Rutherford, NJ, USA, 1:100, lot 5121508, RRID AB_397554) were used as primary antibodies. EnVision FLEX + mouse (linker) (Dako) was use for eNOS staining in mouse samples. EnVision FLEX/HRP (Dako K8000) was used as the secondary antibody for 30 min at room temperature, followed by 3,3'-diaminobenzidine (DAB) staining (Dako liquid DAB + substrate chromogen system, Dako). Sections were then counterstained with hematoxylin, dehydrated, and mounted.

Immunohistochemical stainings were performed with a semiautomated AutoStainer instrument (Lab Vision Corp., Fremont, CA, USA). After deparaffinisation, a heat-induced antigen retrieval in pH 9 was used before incubating the TMA sections with a primary GLP-1R antibody (diluted 1:25, clone 3F52, Developmental Studies Hybridoma Bank/Novo Nordisk A/S, University of Iowa, Iowa City, IA, USA). EnVision FLEX+ Mouse (LINKER) (Dako, Agilent Pathology Solutions, Santa Clara, CA, USA) was used for signal amplification. Antibody binding was visualised with EnVision FLEX kit (Dako). The methods for staining and scoring of Ki-67, somatostatin receptors (SSTR) 1–5 and insulin have been described previously [11 (link)].

IHC reactions were carried out on TMAs using primary antibodies to detect the expression of the proteins. Paraffin sections with a 4 μm thickness were prepared. After deparaffinization and hydration, thermal epitope demasking was performed using a Dako PT Link (Dako, Glostrup, Denmark) apparatus and low pH Target Retrieval Solution (Agilent Technologies, Santa Clara, CA, USA) for 30 min at 97 °C. To visualize the antigen, we used polyclonal rabbit anti-irisin (dilution 1:50; code no. NBP2-14024; Novus Biologicals, Littleton, CO, USA) with EnVision™ FLEX+ Mouse LINKER (Dako). Monoclonal mouse anti-Ki-67 antibodies (ready to use; clone MIB-1; code no. IS626; Dako) and polyclonal rabbit anti-PGC-1α antibodies (dilution 1:3200; code no. NBP1-04676, Novus Biologicals) were used to detect other markers. An automatic DAKO Autostainer Link 48 system (Dako) was used for the IHC reactions and an EnVision FLEX kit (Dako) was used to visualize the antigens. The slides were additionally stained with Mayer’s hematoxylin.

ALK IHC was carried out using the mouse monoclonal antibody 5A4 (Abcam, Cambridge, UK) before 2015, and subsequently using the rabbit monoclonal antibody D5F3 (Roche, Basel, Switzerland). Briefly, 3–4 μm FFPE tumor tissue sections were deparaffinized and incubated in a PT link (Dako, Glostrup, Denmark) with a high pH buffer according to the manufacturer’s recommendations for antigen retrieval. Anti-ALK antibody was then applied for 30 min at 1:50. Slides were incubated at room temperature with EnVision FLEX+ Mouse Linker (Dako) for 15 min. The immune complexes were then detected with the dextran polymer reagent. The percentage of labeled tumor cells and intensity of staining were independently assessed by two pathologists.

Formalin-fixed, paraffin-embedded sections from the above-described 685 tumors were analyzed for claudin-10 protein expression using the Dako EnVision Flex + System (K8012; Dako, Glostrup, Denmark). The claudin-10 antibody was a rabbit polyclonal antibody purchased from Invitrogen (cat # 38–8400; Waltham, MA), applied at a 1:100 dilution following antigen retrieval in LpH buffer (pH 6.0).
Following deparaffinization, sections were treated with EnVision™ Flex + mouse linker (15 min) and EnVision™ Flex/HRP enzyme (30 min) and stained for 10 min with 3′3-diaminobenzidine tetrahydrochloride (DAB), counterstained with hematoxylin, dehydrated and mounted in Toluene-Free Mounting Medium (Dako). Positive control consisted of normal pancreas. In negative controls, the primary antibody was replaced with rabbit serum diluted to the same concentration as the primary antibody.

FFPE tissues were cut into 4.5-µm-thick sections using a microtome and immuno-stained with the monoclonal 5G4 anti-mouse antibody [44 (link)], which labels only the disease-associated αSyn (1:4000; 5 min pre-treatment with 80% formic acid; Roboscreen, Leipzig, Germany). Target retrieval was performed using the DAKO EnVision FLEX Target Retrieval Solution. The DAKO EnVision detection kit, EnVision FLEX peroxidase-blocking solution, 3,3′-Diaminobenzidine chromogen, and EnVision FLEX+ mouse linker (Dako, Glostrup, Denmark) was used to visualize the antibody reactions.
Digital images were obtained with TissueScope^TM LE120 and TissueSnap^TM (Huron, Saint Jacobs, ON, Canada) and cropped using HuronViewer software (Figure S1).