Autostainer universal staining system

All immunostaining was carried out at room temperature by using a DAKO Autostainer Universal Staining System (Autostainer Link 48 DAKO, Glostrup, Denmark). First, sections 4 μm in thickness obtained from selected paraffin-embedded blocks were put on positively charged slides. Second, all the sections were deparaffinized in xylene and dehydrated through a graded series of ethanol solution. Third, antigen retrieval was performed at 96°C (10 mM/L citrate buffer, pH 6) for 40 minutes in a thermostatic bath (PT Link). The sections were incubated with PDL-1 (PDL-1 IHC 22C3 pharmDx, code SK006, DAKO, Glostrup, Denmark) and MHC-1 (HLA-ABC clone W 6/32, code R7000, DAKO, Glostrup, Denmark) for 60 minutes at room temperature. Positive and negative controls were added for each antibody and to each batch of staining. A streptavidin-biotin-enhanced immunoperoxidase technique (K8000 Envision Flex, DAKO, Glostrup, Denmark) in an automated system was used to show immunoreactions. The sections were incubated with DAB and counterstained lightly with hematoxylin to demonstrate binding. Finally, the sections were dehydrated and mounted onto the slides. The tissues known to be positively immunostained were used as positive controls. Normal rabbit serum IgG was used to replace a primary antibody as a negative control.

Slides of routinely processed formalin-fixed, paraffin-embedded [5 ] sections in each case were prepared and stained with antibodies to CD45 (dilution 1:100), CD68 (dilution 1:100) and CD163 (dilution 1:200).
CD45, also known as leucocyte common antigen, is uniquely expressed on the surface of all leucocytes and their progenitor cells [6 (link)]—these include neutrophils, eosinophils, basophils, lymphocytes and monocytes. CD68 is expressed in monocytes and macrophages [7 ]. CD163 was used as an additional stain for cells of monocyte/macrophage lineage and microglia (although the density of microglia was not specifically evaluated in this study) [8 (link)].
Immunoperoxidase staining was performed, following microwave antigen retrieval in citrate buffer at pH6, on an automatic stainer (Dako Autostainer, Universal Staining System) [9 ]. Iron was detected in sections utilising the standard Perl’s staining method [10 ].

Fresh tissue was fixed in 4% neutral buffered formaldehyde and paraffin-embedded. Three micrometer sections were cut on a microtome and stained routinely with haematoxylin-eosin to define representative tumor regions.
Paraffin sections were stained on a Dako Autostainer Universal Staining System (Dako, Denmark). The sections were deparaffinized, and heat-induced epitope retrieval (HIER) was performed by incubation in a buffer solution consisting of 10 mmol/L Tris-base and 0.5 mmol/L ethylene glycol tetraacetic acid, pH 9. After blocking of endogenous peroxidase activity with 5% hydrogen peroxide, the sections were incubated for 60 min with primary antibody against JAM-A/F11R (2E3-1C8, 1 + 400, Sigma-Aldrich, USA). The same antibody clone was used for both cohorts. Detection and visualization of antigen–antibody complex was performed using PowerVision (Novocastra, United Kingdom) and diaminobenzidine (DAB) as chromogen, respectively. Finally, sections were counterstained with Mayers Haematoxylin. Omitting primary antibody and isotype control served as negative controls as well as controls for non-specific staining related to the detection system (Online Resource 1). A tissue microarray consisting of nine different GBMs as well as normal colon, cerebellum, placenta, and rat hippocampus was used as positive/negative control and to monitor inter-run staining variation.

Three μm sections were stained on a Dako Autostainer Universal Staining System (Dako, Glostrup, Denmark). The sections were deparaffinized, and endogenous peroxidase activity was blocked in 1.5% hydrogen peroxide followed by heat-induced epitope retrieval in T-EG buffer for 15 minutes. The sections were then incubated overnight at 4°C with a MMP-2 antibody with affinity towards both latent MMP-2 (72 kDa) and active MMP-2 (66 kDa) (cloneVB3, Merck Millipore, Germany, diluted at 1:300). A polymeric horseradish peroxidase-conjugated system (PowerVision, Novocastra, United Kingdom) was used for detection of antigen-antibody complex followed by visualization with diaminobenzidine (DAB) as chromogen. Mayer’s haematoxylin was used for the nuclear counterstain, and coverslips were mounted with Aquatex. As a negative control, the primary antibody was omitted, showing no staining. Human ovarian carcinoma and bladder tissue were used as positive controls [18 (link), 19 (link)].

Three μm paraffin sections were stained on a Dako Autostainer Universal Staining System (Dako, Glostrup, Denmark). The sections were deparaffinized, and heat induced epitope retrieval (HIER) was performed by incubation in a buffer solution consisting of 10 mmol/L Tris-base and 0.5 mmol/L EGTA, pH 9. After blocking of endogenous peroxidase activity by incubation in 1.5% hydrogen peroxide, the sections were incubated for 60 min with primary antibodies against TfR1 (CD71) (10F11, 1+50, NovoCastra, Newcastle, United Kingdom), FTH (ab65080, 1+800, Abcam, Cambridge, United Kingdom), or FTL (ab69090, 1+1000, Abcam). The antigen-antibody complex was detected using EnVision (Dako). Visualization was performed using DAB (diaminobenzidine) as chromogen. Finally, sections were counterstained with Mayer’s hematoxylin, and coverslips were mounted with Aquatex. Paraffin sections from a TMA containing 28 normal tissues and 12 cancers were used as controls. Omitting primary antibodies served as negative controls as well as controls for non-specific staining related to the detection system.

Mouse cecal tissue, fixed in Bouin’s solution (Sigma) and stored in 70% ethanol, was processed and stained with hematoxylin and eosin by the University of Virginia Research Histology Core [62 , 79 (link)]. Histological scoring for inflammatory infiltration and epithelial cell damage was performed by 2 independent blinded scorers and carried out as previously described [62 , 80 (link), 81 (link)]. Mouse immunohistochemical staining was done using the DAKO Autostainer Universal Staining System with antibody against E. histolytica MIF protein [14 (link)].

The protocol was carried out with the mouse specific HRP/DAB Detection IHC Kit-Micro-polymer (Dako), per the manufacturer's instructions with minor modifications. Antigen retrieval was conducted by heating in a microwave (1,100 W) for 2 min and 10 s in pH 6.0 citrate buffer. Subsequently, the slides were loaded onto a Dako Autostainer-Universal Staining System. Commercially-available anti-rotavirus capsid antibody 2B4, a monoclonal IgG_2B antibody against RVA VP6 (Santa Cruz Biotechnology, Dallas, TX) was used for IHC detection. Additionally, IHC was performed with the anti-dsRNA antibody, clone J2, a monoclonal IgG_2AK antibody that detects dsRNA (Millipore Sigma, Massachusetts, MA). Anti-dsRNA binds to dsRNA 40-bp or longer, independent of their sequence (19 (link)).