3 3 diaminobenzidine dab substrate

Immunohistochemistry (IHC) was performed the same as standard procedures. Briefly, 5 mm tissue sections were deparaffinized in xylene and rehydrated through a gradual decrease in the concentration of ethanol. The antigen epitopes were then unmasked using sodium citrate buffer (pH 6.0) by a standard microwave heating technique. After hydrogen peroxide blocking and normal serum blocking, Ig Blocking Reagent (Vector Laboratories, Burlingame, CA, USA) was used as a blocking buffer. Subsequently, the sections were incubated overnight at 4 °C with the following primary antibodies: CD44 (abcam, ab24504), GFP (cell signaling, #2956), Ki67 (abcam, ab66155), Snail/Slug (abcam, ab180714), CK19 (abcam, ab15463), E-Cadherin (cell signaling, #3195), Vimentin (abcam, ab45939) and N-cadherin (abcam, ab18203). After primary incubation, sections were incubated with the appropriate biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA, USA), followed by incubation with the ABC reagent (Vector Laboratories, Burlingame, CA, USA). Detection was accomplished using DAB (3,3’-diaminobenzidine) substrates (Vector Laboratories, Burlingame, CA, USA). Incubation of sections with phosphate-buffered saline (PBS) served as negative controls. Sections were lightly counter-stained with hematoxylin and mounted. The IHC-stained slides were mounted in Micromount (Leica, Nussloch, Germany).

Extracted femur bones were decalcified while using 14% EDTA (Wako, Tokyo, Japan), embedded into paraffin using standard histologic techniques and sectioned at 5 µm of thickness. The sections were deparaffinized, rehydrated and stained with hematoxylin-eosin (Hematoxylin solution, Sigma-Aldrich, St. Louis, MO, USA; 0.5% Eosin Y, Wako, Tokyo, Japan) for histological analysis. For the immunohistochemistry of mCherry, the antigen retrieval was carried out by using sodium citrate buffer (pH 6.0) with 0.05% Tween 20 for 15 min by a standard microwave heating technique. After cooling down, hydrogen peroxide blocking and Ig blocking were undertaken while using 3% hydrogen peroxide and Ig blocking reagents (Vector Laboratories, Burlingam, CA, USA). After overnight incubation of sections with anti-mCherry, rabbit (Funakoshi, Tokyo, Japan) at 4 °C, the ABC staining kit and DAB (3,3′-diaminobenzidine) substrates (Vector Laboratories, Burlingame, CA, USA) were used for the detection of mCherry, respectively. The sections were counter-stained using hematoxylin and mounted with Micromount (Leica Camera AG, Wetzlar, Germany). The staining was evaluated under light microscopy (FSX100, Olympus, Tokyo, Japan).

The 24-well manual plaque assay is an immuno-plaque method in which vaccine samples and positive controls are pre-diluted based on the sample’s estimated titer before being subjected to a serial dilution and inoculated onto Vero cells that were seeded into 24-well plates prior. Following the inoculation step, samples and positive controls are allowed to incubate for 1 hour at 37°C, at which point the cells are overlayed with growth medium supplemented with methylcellulose and incubated for an additional 6 days. After the incubation, viral plaques are visualized upon cell treatment with serotype-specific anti-dengue monoclonal antibodies (proprietary to Merck & Co., Inc., Rahway, NJ, USA) followed by a horseradish peroxidase (HRP) conjugated secondary antibody (SeraCare Life Sciences, MD, USA). A 3,3’-Diaminobenzidine (DAB) substrate (Vector Laboratories, CA, USA) is then used to illustrate the plaques. Titers of test articles and positive control are determined by plaque counts in wells. This assay was developed following WHO guidelines for plaque-reduction neutralization testing of dengue virus antibodies (34 (link)).

For histology and post-mortem tissues, samples were fixed in 10% Neutral buffered formalin (NBF), paraffin embedded, sectioned at 4 μm and stained with hematoxylin and eosin. For immunohistochemistry, samples were prepared using standard methods. In brief, tissue sections were processed for staining by microwaving in 0.01M citrate buffer, pH 6. After incubation with primary antibodies (Cleaved caspase 3, Cell Signaling, #9664; γH2AX, Millipore #AB5535), samples were incubated with biotinylated secondary antibody (Vector) followed by incubation with Avidin Biotin Complex (Vector); slides were developed in 3,3′-diaminobenzidine (DAB) substrate (Vector) and counterstained in hematoxylin. Tumors and lymphomas images were taken using a Nikon Digital Sight DS-Ri1 camera paired to a Nikon 90i Eclipse microscope. Imaging software was NIS-Elements AR Ver 4.0, 64bit.

All experiments with mice conformed to the protocols approved by the Animal Use Committee of the Second Affiliated Hospital of Chongqing Medical University, and all mice were cared following National Institutes of Health guidelines. For the formation of xenograft tumors, 4- to 5-week-old female BALB/c nude mice (Gempharmatech Biotechnology Co., Ltd., Jiangsu, China) were implanted subcutaneously with sh-NC- or sh-circ_0018289-transduced SiHa cells (5 × 10⁶ cells per mouse) in 200 μL of cell culture medium (n = 6 mice per group). Tumor growth was monitored weekly by caliper measurements and tumor volume was determined by the equation D × d²/2, where D was the longest diameter of the tumor and d was the shortest diameter. Mice were sacrificed at day 35 after cell implantation with CO₂ overdose and the tumors were harvested for weight and expression analysis. Proliferation of tumors was evaluated with paraffin embedded tumor sections (4 μm) by immunohistochemistry using a monoclonal antibody against Ki67 (ab16667, Abcam, dilution 1:200), Vectastain ABC Kit (Vector Laboratories, Burlingame, CA, USA), and 3,3′-diaminobenzidine (DAB) substrate (Vector Laboratories) as described [15 (link)].