K4011

Infected human skin explants treated with Aquaphor or 2% PALA were fixed in Histochoice. Next, paraffin embedded samples were sectioned and stained for HBD2, HBD3, or LL-37. Briefly, dewaxed and rehydrated sections were first subjected to antigen retrieval in sodium citrate buffer using a steamer for 25 minutes. Primary antibody staining was performed at 4 °C overnight in a humidifying chamber. Labelled polymer-HRP anti-rabbit (Dako, K4011), biotinylated secondary goat anti-mouse (EMD Millipore, IHC Select, 20775), and streptavidin-HRP (EMD Millipore, IHC Select, 20774) were used as detection reagents/secondary antibodies. Vina green chromogen (Biocare Medical, BRR807A) was used to visualize protein levels. Aperio AT2 (Leica Biosystems) was used to scan the slides.

Paraffin sections (2 μm) from paraffin-embedded tissue blocks were deparaffinized, re-hydrated, as mentioned above, and antigen recovery was performed using citrate buffer. Slides were rinsed with 1× Tris Buffered Saline (TBS) followed by the blockage of endogenous peroxidase (K4011, Dako) for 10 min. Anti-vWF (1:200 in IHC diluent) was incubated for 3 h and anti-fibronectin (FN; 1:50 in IHC diluent, RE7133-CE, Leica) for 1h, at room temperature. Staining was performed using ABC detection kit (ab93677, Merck Millipore), according to the manufacturer’s protocol. For nuclear staining, slides were immersed in Mayer’s Hematoxylin (RE7164, Leica) for 5 min. Slides were dehydrated and mounted with Entellan (1079610500, Merck) and digitalized using Aperio ImageScope-Pathology Slide Viewing software version 12.4 (Leica, Wetzlar, Germany).

RCC tumors were purchased from Indivumed (Hamburg, Germany). Immunohistochemical staining was performed at Covance Laboratories Inc. (Greenfield, USA), as previously described [30 (link)]. Tumors were fixed in 10% neutral-buffered formalin, embedded in paraffin, and cut into 4 μm sections, which were then dried, deparaffinized, and rehydrated. Heat-induced antigen retrieval was performed for 10 minutes (Antigen Retrieval Solution, Leica, AR9661). Endogenous peroxidase activity was quenched with peroxidase block. Sections were incubated with primary antibody (rabbit anti-human GLS, Abcam, ab156876, 1:200 dilution) for 15 minutes at room temperature. Immunohistochemical reactions were visualized using Dako Rabbit Envision+HRP with DAB+ for use with rabbit primary antibodies (K4011, Dako). Sections were counterstained with hematoxylin.

Paraffin sections (5 µm) of a FACAS patient skin sample and control skin were prepared as explained above, and processed for routine (hematoxylin/eosin) and immunohistological stainings. We used an immunohistochemistry protocol (streptavidin–biotin labeling) employing primary antibodies targeting MPO (MAB3174, R&D Systems) 1:400 overnight at 4 °C or CD163 (ab189915, Abcam) 1:400 overnight at 4 °C. After washing 3 × 5 min with TBS, REAL Detection System Alkaline Phosphatase/RED (K5005, Dako) for MPO staining or labeled polymer-HRP anti-rabbit (K4011, Dako) together with AEC + high sensitivity substrate chromogen ready-to-use (K3461, Dako) for CD163 staining were applied according to the manufacturer’s instructions, and subsequently analyzed by bright-field microscopy. Samples of tonsil tissue served as positive controls. Sections omitting the primary antibody served as negative controls.

Localization of proteins was assessed by immunohistochemistry. Antigens were retrieved using 2100 Antigen Retriever (Aptum Biologics Ltd., Southampton, UK) in Tris–EDTA buffer (pH 9, MKI67) or citrate buffer (pH6). Samples were washed in TBS with 0.1% Tween20 (TBST) (#P1379, Sigma-Aldrich) and blocked for 1 h in 3% BSA in TBST at room temperature (RT). Antibodies against MKI67 (#MIB-1, Dako, diluted 1:500), GNRHR (#19950-1-AP, Proteintech, diluted 1:1000), LHCGR (clone 4G2, antibody donated by Dr Marco Bonomi); supernatant diluted 1:8 (Bonomi et al. 2006 (link)) or FSHR323 (donated by Dr N. Ghinea) (Vannier et al. 1996 (link)) at the concentration of 0.5 µg/mL, were applied on the slides and incubated overnight in 4°C. Endogenous peroxidase activity was quenched by 10-min incubation in 3% hydrogen peroxide (Sigma-Aldrich). Depending on the primary antibody host, Dako EnVision+ System-HRP polymer anti-mouse (K4007, Dako) or anti-rabbit (K4011, Dako) were applied, and visualized with Liquid DAB + Substrate Chromogen System (Dako). Slides were scanned by Pannoramic 250 Slide Scanner (3DHISTECH Ltd., Budapest, Hungary) and images were taken using Pannoramic Viewer (3DHISTECH Ltd.). The percentage of MKI67-stained cells was assessed using ImmunoRatio web application (http://153.1.200.58:8080/immunoratio/) (Tuominen et al. 2010 (link)) from four representative images of each sample.