The remaining biopsies were used for cryo sections using a cryostat (Leica CM1950). Those sections were 4μm for immunostainings and 5μm for quantitative real time polymerase chain reactions (qRT-PCR). Sections for immunostainings were dried for one hour and thereafter stored at -80⁰C. Samples were stained for lymphatic endothelium (Podoplanin and lymph vessels), CD3+ T-lymphocytes and macrophages (CD68). Details of the staining procedures are given in Table 2 . Sections were assessed using a fluorescent microscope (Leica DM 400B). Photos were taken with the Leica DFX345FX camera and LAS software. Counting of lymph vessels was done blinded and manually by two researchers. T-cells and macrophages were analyzed by digital image analyses using Image J. Sections for PCR were used for RNA isolation using the Rneasy microkit (Qiagen, Venlo, The Netherlands) followed by complementary DNA (cDNA) synthesis using the Quantitect kit (Qiagen). This cDNA was used for qRT-PCR with different primer pairs. Used primers were chemokine ligand 2 (CCL2), vascular cell adhesion molecule (VCAM), vascular endothelial growth factor (VEGFC), podoplanin (PDPN) and the housekeeping gene B-actin. Details of the primers are given in Table 3 . PCR was run using the FastStart Universal Sybr green (ROX) master mix (Roche, Basel, Switzerland).
Dm400b
Manufactured by Leica
Sourced in Germany
The DM400B is a compact and versatile microscope designed for a wide range of laboratory applications. It features a high-quality optical system, LED illumination, and various observation methods including bright-field, phase contrast, and differential interference contrast (DIC). The DM400B is a reliable and efficient tool for tasks such as material analysis, biological research, and quality control.
Automatically generated - may contain errors
Lab products found in correlation
Dfc310 fx, by Leica (3 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Cm1950, by Leica (1 mentions)
Quantitect kit, by Qiagen (1 mentions)
Proteinase k, by Agilent Technologies (1 mentions)
Nanozoomer 2 ors, by Hamamatsu Photonics (1 mentions)
Lsr egfr spectrum orange cep 7 spectrum green probe, by Abbott (1 mentions)
Ultraview vox, by Zeiss (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Rabbit anti zo 1, by Thermo Fisher Scientific (1 mentions)
Image it fx signal enhancer, by Thermo Fisher Scientific (1 mentions)
Rabbit anti occludin, by Thermo Fisher Scientific (1 mentions)
Mouse anti claudin 4, by Thermo Fisher Scientific (1 mentions)
Ms 222, by Merck Group (1 mentions)
Histofine simple stain max po, by Nichirei Biosciences (1 mentions)
3 amino 9 ethylcarbazole, by BD (1 mentions)
Lsm 780, by Zeiss (1 mentions)
Oxiselect in vitro ros rns assay kit, by Cell Biolabs (1 mentions)
Carnoy s solution, by Merck Group (1 mentions)
35 protocols using dm400b
1
Immunostaining and qRT-PCR of Lymph Vessels
2
Quantifying Angiogenesis in Granulation Tissue
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Sections (8 µm thick) were fixed with 4% paraformaldehyde, and antigen retrieval was performed using proteinase K (Dako Japan, Tokyo, Japan) according to the manufacturer’s protocol. After 1 h of blocking with PBS containing 10% goat serum, the sections were incubated overnight with mouse anti-pig CD31-FITC antibody MCA1746F (Bio-Rad, Hercules, CA, USA) diluted 1:50 with PBS containing 2% goat serum and 0.05% Triton X-100. Sections were observed at magnifications ranging from 50× to 630× using a fluorescent microscope (DM400B; Leica, Wetzlar, Germany) and photographed with a high-sensitivity digital color fluorescence camera (DFC310 FX; Leica, Wetzlar, Germany) at 20× magnification. CD31-positive vessels were counted using image analysis software (BZ-analyzer). Ten fields under the superficial layer and 10 fields in the middle layer of the granulation tissue were enumerated and used to determine the average number of vessels per 200× magnification “field of view” (FOV).
3
Caspr/paranodin Aggregates Quantification
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The length of immunolabelling of Caspr/paranodin aggregates on cryostat or paraffin sections was measured in the cerebellum and internal capsule. The measurements were performed on images obtained by NanoZoomer 2.O-RS (Hamamatsu) apotome equipped with 20X and 40X objectives or a Leica DM400B optical microscope equipped with 40X, 63X and 100X objectives, according to the chromogen used, respectively fluoroscein, CY3 or DAB. Images acquired from epifluorescence or light microscope were superimposed using ImageJ. The length of paranodin immunostaining was measured inside and outside the plaque in affected and control tissues; the lengths were compared using a variance analysis with Anova.
4
EGFR Amplification Analysis by FISH in GBM
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GBM samples used for FISH analysis were studied using tissue microarrays (TMA). We removed four 0.6-mm cores, from paraffin blocks of each case, corresponding to tumoral areas, confirmed by hematoxylin-eosin staining, using the Beecher Instruments Manual Tissue ArrayerI (Beecher Instruments, Sun Prairie, WI, USA). FISH was carried out using the LSR EGFR Spectrum Orange/CEP 7 Spectrum Green Probe from Vysis (Abbott Laboratories, Downers Grove, IL, USA. Cat. No. 32-191053). Paraffin embedded TMAs were cut into 5-µm sections and these were mounted on Superfrost/Plus microscope slides (Microm International). Hybridizations were performed according to the manufacturer’s instructions. Counterstaining of nuclei was carried out using DAPI.
The fluorescent signal was detected using a photomicroscope, Leica DM400B, equipped with a set of the appropriate filters. Signals were counted in 100–150 non-overlapping tumor cell nuclei in the paraffin sections. The mean signal number for the EGFR gene and CEP 7 was calculated for each case, followed by the calculation of EGFR/CEP 7 ratio. The EGFR gene was quantified as amplified in individual cells when the EGFR/control signal ratio was greater than 2 [9] (link).
The fluorescent signal was detected using a photomicroscope, Leica DM400B, equipped with a set of the appropriate filters. Signals were counted in 100–150 non-overlapping tumor cell nuclei in the paraffin sections. The mean signal number for the EGFR gene and CEP 7 was calculated for each case, followed by the calculation of EGFR/CEP 7 ratio. The EGFR gene was quantified as amplified in individual cells when the EGFR/control signal ratio was greater than 2 [9] (link).
5
Quantitative Analysis of Catecholaminergic Innervation
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The medial PFC subregions were delineated according to plates spanning 14–18 of the mouse brain atlas (Franklin and Paxinos, 2007 ). A 5× magnification was used to trace the contours of the dense TH-positive innervation of the subregions using a Leica DM400B microscope (Figure 3B ). An unbiased counting frame (25 × 25 μm) was superimposed on each contour and counts were made at regular predetermined intervals (x = 175 μm, y = 175 μm) from a random start point. Counting of varicosities was performed at ×100 magnification on 5 of the 12 sections contained within the rostrocaudal borders of our region of interest (Plates 14–18; 1:4 series). A guard zone of 5 μm was used and the optical dissector height was set to 10 μm.
6
Quantifying Celf6 Expression in Mouse Brain
7
Visualizing Tight Junction Proteins in Caco-2 Cells
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When Caco-2 cells had been incubated with the bacteria as described above without FITC-dextran for 11 h at +37°C in 5% CO2, the cells were washed four times with PBS, and then fixed with 200 μl/insert of -20°C methanol for 5 min, followed by an additional four washes with PBS. One drop/insert of Image-iT FX signal enhancer (Life Technologies) was added and the cells were incubated for 30 min at room temperature to block the unspecific binding sites. After washing with PBS, the cells were incubated with 100 μl/insert of rabbit anti-occludin (1:500; Invitrogen), rabbit anti-ZO-1 (1:100; Thermo Scientific), or mouse anti-claudin-4 (1:1000; Invitrogen) antibodies diluted in PBS at room temperature for 1 h, washed four times with PBS, and then incubated with Alexa Fluor 488 goat anti-rabbit IgG (1:1000; Invitrogen) or Alexa Fluor 594 goat anti-mouse IgG (1:1000; Invitrogen) diluted in PBS at room temperature for 1 h. After washing four times with PBS, the Thincert membranes were cut off and mounted on glass slides in Fluoprep medium (BioMérieux). The cells were observed for tight junction proteins using fluorescence microscopy (Leica DM400B).
8
Detecting Muscle Cell Apoptosis via TUNEL
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TUNEL staining was performed to detect muscle cell apoptosis, according to the instructions. After dewaxing and gradient alcohol, the sections were incubated in proteinase K working solution at 37°C in a humidified atmosphere for 15 min. TUNEL reaction mixture (50 μl) was added and incubated for 60 min at 37°C. After rinsed with PBS for 3 times, 50 μl of converter-peroxidase was added to the sections and incubated at 37°C for an additional 30 min, and then rinsed with PBS for three times, incubated with the 100 μl diaminobenzidine substrate. Counterstained with hematoxylin and analyzed by light microscopy (Leica DM400B, Germany) at ×400 magnification. The cells with brown nucleus were defined as apoptotic cells. The percentage of apoptotic cells was calculated as the ratio of the number of TUNEL-positive cells to the total number of cells.
9
Visualizing Zebrafish Neuromast Hair Cells
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FM1-43FX dye (Life Technologies, NY) labeling of hair cells in the neuromasts was performed on live zebrafish larvae (53 (link)). Zebrafish at 5 dpf were immersed in 3 μM FM1-43FX dye in 1X larval water for 45 s and quickly rinsed off. The fish were washed three times with larval water and anesthetized with 0.02% MS-222 (Sigma-Aldrich) for imaging. The numbers of neuromasts of anterior and posterior lateral line were counted. To further evaluate the survival of hair cells in the neuromasts, 5 dpf zebrafish were stained with both FM1-43FX and phalloidin. Briefly, phalloidin staining was performed following FM1-43FX dye treatment and fixation (4% PFA). All nuclei were stained with DAPI. Fluorescence was detected with a confocal microscope (OLYMUS 1000).
DASPEI (2-[4-(dimethylamino)styryl]-N-ethylpyridinium iodide) staining of the neuromast hair cells was performed on live fish. Zebrafish larvae were incubated for 10 min at 28°C in 10 μM DASPEI. The fish were then quickly washed three times with larval water and then transferred into the 2% methyl cellulose for imaging with an inverted fluorescence microscope (DM400B, Leica). The numbers of DASPEI-labeled neuromasts of posterior lateral line were counted.
DASPEI (2-[4-(dimethylamino)styryl]-N-ethylpyridinium iodide) staining of the neuromast hair cells was performed on live fish. Zebrafish larvae were incubated for 10 min at 28°C in 10 μM DASPEI. The fish were then quickly washed three times with larval water and then transferred into the 2% methyl cellulose for imaging with an inverted fluorescence microscope (DM400B, Leica). The numbers of DASPEI-labeled neuromasts of posterior lateral line were counted.
10
Immunohistochemical Detection of Malaria Parasites
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Samples were fixed in 10% neutral buffered formalin, embedded in paraffin and 4 μm-thick sections were cut and stained with standard Hematoxylin-Eosin staining. In order to detect parasites, immunohistochemistry analyses were carried out using a mouse monoclonal anti-Plasmodium HSP70 antibody31 (link) at dilution 1/1000 and a rabbit polyclonal anti-Pfg27 antibody30 (link) at 1/2000. Gametocyte enrichment in different organs was calculated as the ratio of Pfg27-positive cells in Pfg27-stained sections to HSP70-positive cells in HSP70-stained sections observed in 30 fields at 630X magnification for each tissue sample. To determine the location of parasites, indirect labeling of blood vessels was carried out using a rabbit anti-laminin polyclonal antibody (Sigma Aldrich, dilution 1:100) to detect basement membrane of microvasculature. Positive signal was revealed using Histofine Simple Stain MAX PO (mouse or rabbit depending on the primary antibody; Nichirei Biosciences Inc.) according to the manufacturer’s protocol. Color was developed with 3-Amino-9-EthylCarbazole (AEC chromogen; BD Pharmingen). Samples were observed at 630X magnification using a LEICA DM 400B.
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