Subcutaneous transplantation tumours in mice were subjected to immunohistochemistry (IHC) staining using an antigen retrieval method with microwaves. Antibodies against phospho‐Smad3 (p‐Smad3) (Rockland, USA, catalogue 600‐401‐919) and PLK1 (Proteintech, China, catalogue 10305‐1‐AP) were utilized. The antibodies mentioned above were diluted at 1:100. Following that, the sections were washed with PBS, treated with the secondary antibody and visualized using diaminobenzidine. All sections were imaged using a Virtual Slide Microscope (VS120, Olympus, Japan). Staining intensity was assessed in a double‐blind manner using ImageJ v1.8.0 software.
Virtual slide microscope
Manufactured by Olympus
Sourced in Japan
The Virtual Slide Microscope is a digital imaging system that captures high-resolution images of microscope slides. It digitizes entire slides, creating a virtual representation that can be viewed and analyzed on a computer.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Rabbit anti cbs, by Merck Group (1 mentions)
α sma, by Abcam (1 mentions)
Biotin streptavidin hrp based splink detection kits, by ZSGB-BIO (1 mentions)
Fibronectin, by Abcam (1 mentions)
5 protocols using virtual slide microscope
1
Immunohistochemical Analysis of Subcutaneous Tumors
2
Immunofluorescence Staining of Frozen Tissue Sections
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The staining protocols were previously described.30 Briefly, frozen sections were stained with the following primary Abs: goat anti‐mouse TF polyclonal Ab (1:250; R&D Systems), goat anti‐CD31 polyclonal Ab (1:100, R&D Systems), rabbit anti‐α‐ smooth muscle actin (αSMA) Ab (1:100, Abcam), rabbit anti‐collagen type I polyclonal Ab (1:1000, Merck Millipore), rabbit anti‐cytokeratin Ab (1:100, Abcam), and rabbit anti‐caspase3 (Asp175) Ab (1:400, Cell Signaling Technology). Rabbit anti‐caspase3 Ab was reacted at 4°C overnight, and the other primary Abs were reacted at room temperature (RT) for 1 hour. The sections were also stained with secondary Abs including donkey anti‐goat IgG (H+L) cross‐adsorbed secondary Ab Alexa Fluor 647 (1:500) and goat anti‐rabbit IgG (H+L) cross‐adsorbed secondary Ab Alexa Fluor 647 (1:250; Thermo Fisher Scientific) at RT for 1 hour. Hematoxylin‐eosin staining was also carried out. All images were obtained using a Virtual Slide Microscope (Olympus) under identical conditions. Fluorescence image analysis was undertaken by using ImageJ, a public domain Java image‐processing program.
3
Perfusion and Brain Slice Imaging
4
Immunohistochemical Analysis of Tumor Samples
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H&E stained tumor sections were stained using standard histological techniques. Immunohistochemistry was conducted using rabbit-anti-CBS (Sigma-AV45746) 1:4000. Last, slices were photographed with a Virtual slide microscope (Olympus VS120, Japan). The photographs were analyzed with the Image-Pro Plus 6.0 software (Media Cybernetics Inc., Silver Spring, MD, USA). Secondary antibodies: Goat-anti-rabbit IgG (Invitrogen-32460), dilution 1:60.
5
Immunohistochemical Analysis of Kidney Tissue
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Mouse kidney tissues were carefully isolated, fixed with 4% paraformaldehyde, and embedded in paraffin. Then, the paraffin-embedded specimens were sectioned, deparaffinized, and rehydrated with gradient ethanol and subjected to subsequent detections including HE, Masson trichrome staining, and immunohistochemistry. Immunohistochemistry was performed according to the following procedures: the sections were immersed in citric acid antigen repair buffer based on microwave-based antigen retrieval technology as described before [7 (link)]. Then, the sections were reacted with indicated primary antibodies including α-SMA (1 : 100, China, Boster), fibronectin (1 : 100, USA, Abcam) overnight at 4°C. Immediately after that, the sections were rinsed with PBS, incubating with Biotin-Streptavidin HRP-based SPlink Detection Kits (ZSGB-Bio, China), and color was developed with DAB. Finally, Counterstaining was performed with hematoxylin. Signals were detected with a Virtual Slide Microscope (VS120, Olympus, Japan).
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