Dako envision kit

Details of the procedures used have been described in our previous study [16 (link)]. Briefly, 4 μm sections were deparaffinized in xylene and rehydrated in a descending ethanol series. After antigen retrieval in 10 mM sodium citrate (pH 6.0), endogenous tissue peroxidase activities were quenched by 3% hydrogen peroxide for 20 min followed by blocking with 5% normal goat serum (Dako, Glostrup, Denmark) for 1 hour. The sections were immunostained with rabbit monoclonal anti-P4HB (at 1:100 dilution; Cell Signalling Technology Inc., Danvers, MA, USA), rabbit monoclonal anti-PECAM-1/CD31 (1:50 dilution; Abcam^®, Cambridge, MA, USA), and rabbit monoclonal anti-VEGF antibodies (both at 1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. After incubation with horseradish peroxidase (HRP)-conjugated antibody (Invitrogen-Zymed Laboratories, South San Francisco, CA, USA) at 1:200 dilution, signal was detected using a ready-to-use DAKO EnVision™+ Kit (Dako). Nonspecific immunoglobulin was substituted as negative controls.

Paraffin-embedded tissue samples were cut into 4 μm-thick sections and mounted on polylysine-coated slides. Samples were dewaxed in xylene and rehydrated using a graded series of ethanol solutions. After deparaffinization, endogenous peroxidase activity was blocked by incubation in a 3% peroxide-methanol solution at room temperature (RT) for 10 mins, and then antigen retrieval was performed at 100°C in an autoclave for 7 mins. Samples were then incubated at RT for 30 mins. Afterwards, sections were washed with phosphate-buffered saline (PBS) three times for 5 mins each time. They were then incubated with mouse anti-human CD163 antigen monoclonal antibody (clone 10D6, Zhongshan Goldenbridge Biotechnology Co., LTD., Beijing, China), mouse anti-human MMP9 antigen monoclonal antibody (clone Sc-2c3, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-human CD34 antigen monoclonal antibody (clone QBEnd/10, Zhongshan Goldenbridge Biotechnology Co., LTD., Beijing, China). Thoroughly washing with PBS was then performed, and primary antibody binding was visualized using a DAKO EnVision kit (DAKO, Glostrup, Denmark) following the manufacturer's instructions. Finally, sections were faintly counterstained with hematoxylin and mounted with glycerol gelatin.

Tissue sections (4 μm) were stained with hematoxylin and eosin for histological analysis and with specific primary anti-human antibodies against NXF3 (1:150; LS-C31687; LifeSpan Biosciences, Inc., Seattle, WA, USA) for IHC. Following microwave antigen retrieval, the tissues were incubated with the primary antibodies overnight at 4°C followed by a 30-min incubation with the secondary antibody (Dako EnVision kit, Dako, Glostrup, Denmark). The reaction was visualized with diaminobenzidine and the tissues were counterstained with hematoxylin.
The tissue sections were viewed at ×200 magnification using a Leica DMI6000B inverted microscope (Leica Microsystems, Heidelberg, Germany) and images were captured. Two experienced pathologists independently assessed all IHC staining. The scoring for nuclear NXF3 expression was based on the staining proportion and intensity. The staining proportion was scored as follows: 0–25% staining, 1; 26–50% staining, 2; 51–75% staining, 3; and 76–100% staining, 4. The staining intensity was scored as follows: Negative intensity, 0; weakly positive, 1; moderately positive, 2; and strongly positive, 3, according to a previous study (12 (link)). The sum of the proportion and intensity scores was used to calculate the final staining score, which was then categorized as low (1 (link)–5 (link)) or high (6 (link)–7 (link)).

Paraffin-embedded sections (4 μm thick) of subcutaneous AT were deparaffinized in xylene and rehydrated through descending grades of ethanol (100%, 95%, and 75%) to water. Antigen retrieval was performed by placing slides in a target retrieval solution (pH 6.0; Dako, Glostrup, Denmark) in a pressure cooker, boiling for 8 min, and cooling for 15 min. After washing in PBS, endogenous peroxidase activity was blocked with 3% H₂O₂ for 30 min, and nonspecific antibody binding was blocked with 5% fat-free milk for 1 h, followed by 1% bovine serum albumin solution for 1 h. The slides were incubated at room temperature overnight with primary antibody (1:100 dilution of rabbit polyclonal anti-Caveolin-1 antibody; Cell Signaling Technology #3238s). After washing with PBS (0.5% Tween), slides were incubated for 1 h with a secondary antibody, namely, goat anti-rabbit conjugated with horseradish peroxidase (HRP) polymer chain Dako EnVision Kit from (Dako, Glostrup, Denmark) and color was developed using 3,3′-diaminobenzidine (DAB) chromogen substrate. Specimens were washed, counterstained, dehydrated, cleared, and mounted, as described elsewhere [25 (link)]. For analysis, digital photomicrographs of the entire AT sections (20X; PannoramicScan, 3DHistech, Hungary) were used to quantify the immunohistochemical staining using ImageJ software (NIH, Bethesda, MD, USA).

In order to analyze the production of NP extracellular matrix components in 3D transplants, the grafts were dissected into 4 parts, embedded in TissueTek (Sakura Finetek, Staufen, Germany), frozen in liquid nitrogen and stored at -80°C until sectioning. Cryosections with a thickness of 6 μm were prepared. To demonstrate proteoglycan formation, Safranin O staining was conducted. The samples were incubated for 30 min with a 0.7% Safranin O solution (Sigma-Aldrich) and subsequently counterstained with a 0.2% Fast green solution (Sigma-Aldrich). For the immunochemical detection of collagen type II, a primary monoclonal rabbit-anti-human antibody (Acris Antibodies, Herford, Germany) was used. The detection was performed using the DAKO EnVision Kit (DAKO, Hamburg, Germany) according to the manufacturer’s protocol. In brief, the cryosected transplants were incubated with primary antibody solution for 40 min. The secondary antibody (horseradish peroxidase labeled goat-anti-rabbit antibodies) solution was also applied for 40 min. Finally, the cryosected transplants were incubated with the substrate AEC for 10 min and then counterstained with hematoxylin (DAKO) for 10 minutes. Pictures of both stainings were taken using an Olympus CX41 microscope and an Olympus Colorview camera (Olympus Soft Imaging Solutions GmbH, Hamburg, Germany).