Ultravision quanto detection system

The tumor microarrays (TMAs) were constructed from formalin-fixed, paraffin embedded surgical specimens, and CLEC3B staining was performed with UltraVision Quanto Detection system (Thermo scientific, California, US) following the protocol recommended and hematoxylin was used for counterstaining. The images were obtained by Nikon eclipse Ti-s microscope (Tokyo, Japan) and assessed by two investigators who had no knowledge of the patients’ clinical data to exclude subjectivity. For IHC results assessment, a previous scoring method was used [31 (link)]. Composite expression score (CES) with full range from 0 to 12 was performed to show the staining intensity and frequency of positive cells.

After 14 days of culture, the cell-laden hydrogels were washed with PBS and fixed with 10% neutral formalin for 2 days. After that, a tissue processor (Leica ASP300 S, Heidelberg, Germany) was used to dehydrate the samples, followed by paraffin fixation. Then, the embedded samples were sectioned to obtain cross-sections 4 µm thick. The cross-sections were deparaffinized, stained with Haemotoxylin and Eosin (HE) stainning, and examined using a microscope. In addition, the immunohistochemical expression of MMP2, MMP9, and collagen I were analyzed for cellular proliferation quantification. Briefly, the tissue section was deparaffinized and rehydrated with xylene and a series of ethanol solutions. The tissue sections were treated for 15 min with a hydrogen peroxide block and protein block solutions from the UltraVision Quanto Detection System (Thermo Fisher Scientific). The slides were then washed three times with TBST and incubated with a primary antibody amplifier and HRP polymer (UltraVision Quanto, Thermo Fisher Scientific) for 10 min. Finally, the sections were stained using 3’-Diaminobenzidine (DAB) and then counter-stained with hematoxylin, dehydrated, and examined under a microscope. All images from the microscope were taken with a built-in camera (BX-53; Olympus, Tokyo, Japan).

Paraffin-embedded human glioma tissue microarrays with five normal brain tissue were purchased from Shanghai Outdo Biotech and Pantomics, Inc. UltraVision Quanto Detection System (Thermo Fisher Scientific Inc.) was used for immunohistochemistry. Arrays were incubated with anti-DSE antibody (1:100) in 5% bovine serum albumin/PBS 16 hours at 4°C. The specific immunostaining was visualized with 3,3-diaminobenzidine and counterstained with hematoxylin (Sigma). Images were obtained by TissueFAX Plus Cytometer. The staining index was calculated as previous descripted [16 (link)]. A final score ≥ 6 were be considered high expression.

Femur sections were subject to IHC staining for Ki-67, BMP-2 and osteocalcin to detect the ability of bone formation. UltraVision Quanto Detection System (Thermo Scientific, Fremont, CA, USA) was used according to the manufacturer’s instructions. Briefly, tissue sections were incubated with UltraVision Hydrogen Peroxide Block for 10 min to stop endogenous peroxidase activity, and then UltraVision Protein Block to block nonspecific protein. The monoclonal antibodies used for immunohistochemistry were anti-Ki-67 (1:100), anti-BMP-2 (1:100) and anti-osteocalcin (1:100). Antibodies were added and incubated at 4 °C overnight. Subsequently, sections were incubated with Primary Antibody Amplifier Quanto for 10 min and then HRP Polymer Quanto for 10 min. Visualization of the specific binding was developed using DAB/Chromogen. Sections were then mounted, cleaned, cover-slipped and quantified, analyzed using a QuPath (https://qupath.github.io, accessed on 22 March 2020) the color and background estimates were applied for each IHC analysis for stain separation within QuPath using color deconvolution. By selecting a representative area containing an area of background along hematoxylin and DAB staining.

The human HNSCC tissue microarrays (paraffin‐embedded), including follow‐up survival information, were provided by SuperBioChips. The arrays underwent incubation with anti‐CUL4B antibody (1:200) in 0.1% Triton X‐100 and 5% bovine serum albumin/PBS (Sigma) for sixteen hours at 4°C. Then, the fixed cells were incubated in 0.1 mol/L Tris‐HCl, pH 7.5, containing 0.05 mol/L acetic acid (chondroitinase reaction buffer) and 0.5 units/mL protease‐free chondroitinase ABC (Sigma) for two hours at 37°C. After three washes, the cells were incubated in blocking solution and stained with 1B5 antibody (1:20) and Alexa 488‐conjugated goat anti‐mouse IgG (1:100, Thermo Fisher Scientific Inc, Waltham, MA, USA). The immunostaining was observed using 3,3‐diaminobenzidine and counterstained with hematoxylin (Sigma) using the UltraVision Quanto Detection System (Thermo Fisher Scientific Inc). The distribution and positive intensity of CUL4B were analyzed by two individuals in a double‐blind experimental design. The staining scores were assigned according to the percentage of positive tumor cells (0, 0%; 1, <25%; 2, 25‐50%; 3, 51‐75%; and 4, >75%) and staining intensity (0, none; 1, weakly stained; 2, moderately stained; and 3, strongly stained).