Further details on the fluorescent immunohistochemistry (fIHC) are provided in Additional file 2 . Tumors were collected for fIHC analyses 20–22 h after daily RO4987655 administration for each [18F] FDG-PET time point (days 0, 1, 3 and 9). For fIHC, tumor samples were fixed by immersion for 24 h in a solution of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) containing phosphatase inhibitors. Tissue blocks containing 4 to5 tumor samples of different treatment groups were snap frozen, cryosectioned at 14-μm thickness, and thaw-mounted on Superfrost Plus slides. Single- and multi-labeling experiments were performed as described previously [27 (link)]. Briefly, sections were incubated with rabbit anti-GLUT1 (Abcam, AB653, dilution 1:300) and anti-hexokinase 1 (Abcam, AB65069, dilution 1:200) antibodies and were visualized by incubation with Alexa488 conjugated donkey anti-rabbit antibody. Whole slide images were captured on a Metasystems Vslide system (Newton, MA, USA) equipped with appropriate filter sets using a × 10 objective. Channel grey scale images were analyzed using ImageJ software (1.45p NIH, Bethesda, MD, USA) and GLUT1 and hexokinase 1 fluorescence intensity were measured from 50 randomly selected spots within each tumor sample. Supporting quantification figure is available as an additional information (Additional file 3 ).
Vslide system
Manufactured by MetaSystems
Sourced in United States
The VSlide system is a versatile lab equipment designed for various research and analysis applications. It provides a compact and efficient platform for slide-based experiments and observations. The core function of the VSlide system is to enable the precise positioning and manipulation of slide specimens for microscopic examination or other experimental purposes.
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3 protocols using vslide system
1
Quantitative Fluorescent Immunohistochemistry for GLUT1 and Hexokinase 1
2
ADAM33 Expression Analysis in Tissues
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The tissue microarray (TMA) blocks were serially sliced to generate 5-μm-thick sections. The sections were deparaffinized in xylene using two changes for 10 minutes each. Hydrate sections gradually through graded alcohols: wash in 100% ethanol three times for 1 minutes each, and 80% ethanol three times for 1 minutes each. The endogenous peroxidase activity was blocked using an Advance kit (Dako), with 5% hydrogen peroxide in methanol. The sections were then incubated with the primary antibody anti-ADAM33 at a dilution of 0.2 μg/mL overnight at 4 °C. They were then incubated with the secondary antibody (Dako Advance HRP System, DakoCytomation, Inc., USA) for 30 min, which was followed by incubation with 3,3′-diaminobenzidine and hydrogen peroxide substrate (DakoCytomation, Inc., USA) for 3 min to visualize positive staining. Finally, the sections were counter-stained in Harris hematoxylin. The staining procedures included a negative control (without primary antibody) and a positive control (normal lung tissue). The images were obtained using a motorized Axio Imager Z2 microscope (Carl Zeiss, DE), equipped with an automated scanning VSlide system (Metasystems, DE).
3
Microscopic Screening for Hemoplasmas
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Blood smears were prepared from EDTA K2 anticoagulated blood within five minutes after blood collection to ensure no hemoplasma detachment from red blood cell surface [28 ]. The slides were stained using Romanowsky staining (May-Grünwald - Giemsa) in an automated stainer (Sysmex XE-2100, Sysmex Corporation, Japan) to ensure constant staining quality. Blood smear preparations were scanned for the presence of hemoplasmas using a motorized Axio Imager Z2 microscope (Carl Zeiss, Jena, DE) equipped with an automated scanning VSlide system (Metasystems, Altlussheim, DE) at the Multiuser Conventional and Confocal Fluorescence Microscopy Laboratory at the Department of Biological Sciences, Federal University of Paraná State, Brazil. For each slide, 1000 red blood cells were counted in different randomly chosen high power (100x) fields and the number of structures morphologically compatible with hemotropic mycoplasmas noted. Microscopy results for detection of hemoplasmas were subsequently compared with molecular PCR results.
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