IHC staining was performed using paraffin-embedded tumor sections. After deparaffinization, rehydration, and high-temperature antigen retrieval, tumor sections were incubated with specific antibodies against PCNA (Santa Cruz Biotechnology, #sc-56) or cleaved caspase-3 (Cell Signaling Technology, #9661S) and then subjected to horseradish peroxidase–linked secondary antibodies for 1 hour at room temperature. The DAB substrate kit (Servicebio, #G1212-200T) was applied for visualizing staining signals. Representative IHC images were captured with a digital pathology scanner (Leica) at the ×400 magnification.
Digital pathology scanner
Manufactured by Leica
The Digital pathology scanner is a high-resolution imaging device designed for the acquisition and digitization of tissue samples. It captures detailed images of microscopic samples, enabling pathologists to analyze and diagnose medical conditions more efficiently.
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4 protocols using digital pathology scanner
1
IHC Staining of Tumor Sections
2
Immunohistochemical Analysis of OSCC
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Tissue sections were repaired by sodium citrate under high pressure. Porphyromonas gingivalis was detected using polyclonal Rabbit RgpB antibody (1 : 50, Biorbyt, orb51295, Cambridge, UK). Ki67 (1 : 200, CST, 12202T, Boston, MA, USA) was used to show the proliferation of OSCC. MUC1 (1 : 200, Proteintech, 23614‐1‐AP, Wuhan Sanying, Wuhan, Hubei, P.R.C) and Caspase 3 (1 : 200, Proteintecch, 19677‐1‐AP, Wuhan Sanying, Wuhan, Hubei, P.R.C) were used to detect the expression level of MUC1 and Caspase 3. A Leica digital pathology scanner was used to analyze the images. Optical density values were analyzed using aperio imagescope software (Aperio, Vista, CA, USA). Immunohistochemical staining intensity was scored according to four values in excel : Total Intensity of Strong Positive, Total Intensity of Positive, Total Intensity of Weak Positive and Area; the formula is (3 × Total Intensity of Strong Positive) + (2 × Total Intensity of Positive) + (1 × Total Intensity of Weak Positive)/Area (in units per μm2) and the resulting value is the average optical density value of the selected area for each sample. Before statistical analysis, the data need to be homogenized, scaled up or down by equal proportions, and checked that the maximum value does not exceed 100 or 300. All procedures could refer to our previous work [28 (link)].
3
Immunofluorescence and Immunohistochemistry Protocols
4
Protein Expression and ROS Detection
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Paraffin-embedded tissue samples underwent deparaffinization and antigen retrieval. The primary antibodies were PCNA (1:10000, CST, 13,110), HMGB1(CST, 6893 S), CRT (CST, 12,238 S), Caspase 3 (1:200, Proteintecch, 19677-1-AP). Live/Dead stain kit (Calcein acetoxymethyl ester and propidium iodide probes) and the probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA) used to detect the ROS level were performed strictly following the instructions. IHC images were obtained with a digital pathology scanner (Leica), and IF images were taken with a confocal microscope (Leica).
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