Cells were seeded at a density of 5×104 cells per well of a 24-well plate, 24 hours prior to 0.5–50.0 µM RHPS4 exposure for 72 hours. Alamar Blue assay (Invitrogen, UK) was conducted according to the manufacturer guidelines and fluorescence emission measured at 585 nm using a plate reader (Tecan, Switzerland). Percentage viability was calculated related to vehicle-only treated controls. Qualitative images of RHPS4-treated brain tumor cells were taken using a standard light microscope (Leica, UK). IC50 values refer to the concentration at which the tumor population viability is 50% that of the corresponding untreated tumor population.
Standard light microscope
Manufactured by Leica
Sourced in United Kingdom, United States, Germany
The Standard light microscope is a versatile optical instrument used to magnify and observe small objects. It utilizes visible light and a system of lenses to enlarge the image of a specimen, allowing users to examine its details more closely. This microscope is a fundamental tool in various fields, including biology, materials science, and medical research.
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6 protocols using standard light microscope
1
RHPS4 Cytotoxicity in Brain Tumors
2
Comprehensive Tissue Analysis Protocol
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Sections (4-μm thick) of embedded primary tumor, heart, liver, spleen, lung, and kidney were stained for a routine histological examination and morphometric analysis. The brain slices were immunostained with the Ki67 (1:400), phospho-JNK (1:100), and cleaved caspase-3 (1:100) antibodies. The digital images were captured under a standard light microscope (Leica).
3
Tissue Microarray Immunohistochemistry for RASSF6
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A tissue microarray was used in this study. Immunohistochemistry staining was performed, as described previously [22 (link)]. For antigen retrieval, tissue sections were boiled in 0.01 M citrate solution (pH 6.0) and incubated with primary antibody to RASSF6 (1:150, Sanying, Wuhan, China). Tissue sections were observed with a standard light microscope (Leica).
4
Histomorphometric Analysis of Bone Formation
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After micro‐CT scanning, the specimens were dehydrated in a graded ethanol series (70–100%) and then embedded in a methylmethacrylate solution that was polymerized at 37 °C within 1 week. Afterward, thin sections (≈50 µm in thickness) were prepared using the modified interlocked diamond saw (Leica Microtome, Wetzlar, Germany) and stained with 1.2% trinitrophenol and 1% acid fuchsin (Van‐Gieson staining). Bone formation was qualitatively measured with a standard light microscope (Leica) equipped with a digital image analysis system (Image‐Pro Plus software, Media Cybernetics, Silver Spring, USA). Prior to histomorphometric analysis, bone and material were pseudocoloured using Adobe Photoshop 6.0 and then measured using a digital image analysis system (Image‐Pro Plus software, Silver Spring, USA). The bone volume fraction (ratio of bone tissue area to the implant pore area within the implant) was calculated based on Van‐Gieson staining and compared statistically.
5
Bone Marrow Histopathology Analysis
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Three-µm-thick sections from Bouin’s solution-fixed, paraffin-embedded BM biopsies were stained with haematoxylin-eosin and immunostained with an automated stainer device (Ventana-Ultra, Ventana Medical Systems, Tucson, AZ, USA) using polyclonal antibodies against CD34 (clone QBEnd/10; #NCL-L-END; Novocastra; Leica Microsystems, Milton Keynes, UK) at a 1:50 dilution at 37 °C for 36 min, and CD68 (clone PG-M1, cod. M0876; Dako, Carpinteria, CA, USA) at a 1:50 dilution at 25 °C for 30 min. Sinusoid-like vessel density and CD68+ cellular extensions were analyzed in 10 random high power fields employing 40× magnification using a standard light microscope (Leica, Wetzlar, Germany).
6
BMP5 Immunohistochemistry on Tumor Microarrays
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BMP5 immunohistochemistry was performed on tumor microarrays (TMAs) from Fanpu Biotech, Inc., and the pathological information can be found in Additional file 1 : Table S4. For antigen retrieval, tissue sections were boiled in 0.01 M citrate solution (pH 6.0) and incubated with primary antibody to BMP5 (1:200, Proteintech, China). Tissue sections were observed with a standard light microscope (Leica). The intensity of staining ranged from negative (0) to weak positive (1–2) or strong positive (3).
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