Four‐week‐old female BALB/c‐nu/nu mice (obtained from the Laboratory Animal Center of SUN YAT‐SEN University) were kept in the Animal Room F zone of Zhongshan School of Medicine; 1 × 106 CFPAC‐1 or PANC‐1 cells with 2 × 106 CAFs#1 cells in 150 μL PBS were inoculated subcutaneously into the left flank of nude mice. Tumors were measured every 3 days. Tumor volume was calculated with the formula: V = 0.5 × length × width 2. The tumor‐bearing mice were killed 33 days after inoculation, and the tumors were subsequently removed for further study. Tumor slices were stained using a Masson Trichrome Staining Kit (LEAGENE, #DC0033). Digital microphotographs (200× and 400×) were taken using a NIKON Eclipse Ci microscope, and three microscopic fields were randomly selected. The images were analyzed using ImageJ software (version 1.8), and the average stromal proportions were calculated. The detailed operating protocol using ImageJ software can be found in Section 2.5 .
Eclipse ci microscope
Manufactured by Nikon
Sourced in Japan, United States, Hungary, United Kingdom, Germany
The Eclipse Ci microscope is a high-quality optical microscope designed for a variety of laboratory applications. It features a stable and ergonomic design, providing reliable performance and user comfort. The microscope offers a range of optical capabilities, including brightfield and phase contrast observation, to support a wide variety of sample types and research needs.
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Lab products found in correlation
Image pro plus 6, by Media Cybernetics (7 mentions)
Digital sight ds fi2 camera, by Nikon (6 mentions)
Ds fi3 camera, by Nikon (5 mentions)
Nis elements software, by Nikon (5 mentions)
Oil red o, by Merck Group (4 mentions)
Ds ri1 camera, by Nikon (3 mentions)
Ds fi2 camera, by Nikon (3 mentions)
Ds ri2 camera, by Nikon (3 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (3 mentions)
Nis elements imaging software, by Nikon (3 mentions)
Microm hm 355, by Thermo Fisher Scientific (3 mentions)
Ds vi1 camera, by Nikon (3 mentions)
Technovit 7100 resin, by Kulzer (3 mentions)
Sk 4100, by Vector Laboratories (2 mentions)
Chemray 240, by Rayto (2 mentions)
Chemray 800, by Rayto (2 mentions)
Liquid dab substrate chromogen system, by Agilent Technologies (2 mentions)
Rnascope zz probe technology, by Advanced Cell Diagnostics (2 mentions)
Rnascope 2.5 hd reagents red kit, by Advanced Cell Diagnostics (2 mentions)
Donkey serum anti mouse igg, by Thermo Fisher Scientific (2 mentions)
174 protocols using eclipse ci microscope
1
Evaluating Tumor Stromal Proportion in Xenograft Model
2
Sporulation and Tetrad Dissection Protocol
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Yeast strains used in the present study are listed in Supplementary Table S1. If not stated otherwise BY4741 and BY4742 are the parental strains. Sporulation and tetrad dissection of Δgrx3/4 diploids were performed according to published methods [25 (link),26 (link)]. Briefly, GRX3/Δgrx3 GRX4/Δgrx4 diploid strains were grown on YPAD agar at 30°C for 2–3 days. A swab of cells was patched onto pre-spore media (0.8% yeast extract, 0.3% peptone, 2% agar and 40% glucose) and grown at 30°C for 2 days. Further, cells from pre-spore plates were patched on to sporulation media composed of 0.25% yeast extract, 1.5% potassium acetate, 2% agar, 0.05% glucose, and 1× spore amino acids (Supplementary Table S2) and grown at 30°C. Diploid cells sporulated in 4–5 days and were then subjected to tetrad analysis. For tetrad dissection, cells were scraped from sporulation plate using a sterile stick, and gently suspended in 40 µl of zymolyase (1 mg/ml in 1 M sorbitol) and incubated for 15 min at 30°C. The cells were diluted gently by adding 500 µl of sterile deionized water. Using a sterile wire loop, the cell suspension was mixed gently and a loopful of cells was spread in a line on a YPAD plate. The tetrads were dissected using Nikon eclipse Ci Microscope. Crosses of haploid strains were performed by micromanipulation of zygotes [25 (link)].
3
Morphological Effects of Macrophage Conditioned Media
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After 72 h of interaction between CMs obtained from culture of M0, M1 and M2 macrophages, morphological abnormalities in hGCs and COV434 cells were evaluated by Giemsa stain [19 (link)]. hGCs and COV434 cells were seeded in a 24-well plate with round coverslips at densities of 7.0 × 105 and 3.5 × 105 cells/well, respectively. After adherence, 500 μL of CMs of M0, M1 and M2 were added to the cells and incubated for 72 h at 37 °C with 5% CO2 atmosphere. After incubation, the CMs were removed, cells were washed twice with PBS, and fixed with cool methanol for 10 min at room temperature (RT). Then, the methanol was removed, cells were washed again twice with PBS, Giemsa solution in H20 (1:10) was added, and the cells were incubated for a further 30 min. After incubation, the cells were washed several times with tap water and the coverslips were removed, dried, and mounted with DPX medium. Cells were observed under the Eclipse CI microscope (Nikon, Japan).
4
Immunohistochemical Analysis of Tissue Samples
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Tissues were fixed in formalin overnight, then transferred to 70% ethanol, and embedded in paraffin (ARUP histology core). Formalin-fixed paraffin-embedded sections (4 micron) were used for hematoxylin and eosin and IHC staining. Antigen retrieval was performed by boiling slides for 20 min in 0.01 M citrate buffer, pH 6.0. Slides were blocked for 15 min with 3% H2O2, followed by 5% goat serum in PBS containing 0.1% Tween-20 (PBST). Primary antibodies were incubated overnight at 4 °C and include the following: BCL2 (#M088701-2, clone 124, Agilent), MYC (ab32072, Abcam), NEUROD1 (ab205300, Abcam), and ASCL1 (#556604, BD Pharmingen). Slides were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Vector Laboratories, 1:200) and developed with DAB (Vector Laboratories). A Nikon Eclipse Ci microscope and DS-Fi3 camera were used for imaging.
5
Histological Evaluation of Lung Tissue
6
Immunohistochemistry of PDAC Tumors
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4-μm FFPE sections from PDAC tumors were deparaffinized and treated with 1% H2O2. Antigen retrieval was performed using citrate buffer (pH 6.0) for all antibodies, except for MUC5B and HLA-DR (for which Tris-EDTA buffer (pH 9.0) was used). Slides were blocked with 10% normal horse serum (Vector Labs, S-2000) and the antibodies listed in Supplementary Data 12 were used. Visualization was achieved with 3,3’-diaminobenzidine as a chromogen (Vector Labs, SK4100). Counterstaining was performed with Mayer hematoxylin (Sigma Aldrich, MHS16). Images were taken with a Nikon Eclipse Ci microscope (Fig. 1a ) or scanned by the Pannoramic SCAN II scanner, with 20×/0.8 objective (3DHISTECH, Budapest, Hungary) (Fig. 2b, c ).
7
Hematoxylin-Eosin Tissue Staining
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The 4% PFA-fixed tissues were embedded in paraffin and 4 μm thick sections were prepared. Section samples were de-paraffinized and re-hydrated. Samples were treated with hematoxylin, eosin, a range of concentrations of alcohol, and xylene to complete HE staining. All images were observed and captured under an ECLIPSE Ci microscope (Nikon, Tokyo, Japan).
8
Quantitative Analysis of Vaginal Extracellular Matrix
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The vaginal tissue was fixed in 10% formalin for 24 h, embedded in paraffin, and cut into 5-μm-thick sections. The vaginal tissue sections were stained following the standard procedures for Masson trichrome staining, sirius red staining, and Verhoeff-van Gieson staining. For image analysis, the slides were viewed under a Nikon Eclipse CI microscope, and images of five randomly selected fields per slide were captured with a Nikon DS-U3 camera. Image-Pro Plus computer software was used to calculate the percentage of collagen I and collagen III (sirius red stain, ×200 magnification). The percentage of elastin in the lamina propria was analyzed using National Institutes of Health ImageJ software (Verhoeff-van Gieson stain, ×400 magnification).
9
Cannabinoid Effects on Behavior and Histology
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Mice were administered vehicle, 50 mg/kg THC, 30 mg/kg JWH-073, or 1 mg/kg AM-2201 daily for 4 days at ∼9 am, and latency to tail withdrawal and body temperature were measured before the first and 60 min after each injection. On day 5, ∼24 h after the previous injection, each mouse was observed for 10 min before and 10–30 min after IP injection of 10 mg/kg SR141716A. The number of times each animal exhibited the following behaviors was counted: rear, scratch, groom, forepaw tremor, retropulsion, and head shake.
Within 1 h of the end of the aforementioned experiment, mice were euthanized and necropsied. Heart, lungs, thymus, liver, kidneys, spleen, brain, and skeletal muscle (right gastrocnemius) were collected and stored in 10% neutral buffered formalin. Postfixation, tissues were placed into cassettes (Simport). For the heart, a longitudinal section through both ventricles from base to apex created two halves and permitted visualization of ventricles and atria. For the brain, four coronal sections, anterior to posterior, were submitted. Routine histological processing was performed by the Histology Laboratory of the College of Veterinary Medicine at North Carolina State University. Slides with 5 μm-thick hematoxylin and eosin stained sections of the organs collected were evaluated by light microscopy (n=4–9) using a Nikon Eclipse Ci microscope.
Within 1 h of the end of the aforementioned experiment, mice were euthanized and necropsied. Heart, lungs, thymus, liver, kidneys, spleen, brain, and skeletal muscle (right gastrocnemius) were collected and stored in 10% neutral buffered formalin. Postfixation, tissues were placed into cassettes (Simport). For the heart, a longitudinal section through both ventricles from base to apex created two halves and permitted visualization of ventricles and atria. For the brain, four coronal sections, anterior to posterior, were submitted. Routine histological processing was performed by the Histology Laboratory of the College of Veterinary Medicine at North Carolina State University. Slides with 5 μm-thick hematoxylin and eosin stained sections of the organs collected were evaluated by light microscopy (n=4–9) using a Nikon Eclipse Ci microscope.
10
Immunohistochemical Quantification of ROR-γt and TNF-α
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Five-micrometer sections were immune-stained for ROR-γt and TNF-α. following endogenous peroxidase activity quenching in 3% hydrogen peroxide (Dinâmica, Diadema, SP, Brazil). Antigen retrieval (AR) was performed in boiling citrate buffer (pH 6.0). The primary antibody was incubated overnight at 4°C, followed by EnVision HRP and Envision+ (K1491; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) at 37°C for one hour. The sections were then stained with 3,3′-diaminobenzidine tetrahydrochloride (DAB, Dako; Agilent Technologies, Inc.) for five min at 37°C and counter-stained with hematoxylin.
ROR-γt and TNF-α expression was evaluated by inflammatory positive cell counting within the subepithelial connective tissue over 10 fields per case at magnification, ×400 (×40 objective lens, field diameter of 0.44 mm) using a CCD camera on a Nikon Eclipse Ci microscope. Epithelial cells were not taken into account for ROR-γt and TNF-α expression. The individual who performed the counting was blind to the experimental groups.
ROR-γt and TNF-α expression was evaluated by inflammatory positive cell counting within the subepithelial connective tissue over 10 fields per case at magnification, ×400 (×40 objective lens, field diameter of 0.44 mm) using a CCD camera on a Nikon Eclipse Ci microscope. Epithelial cells were not taken into account for ROR-γt and TNF-α expression. The individual who performed the counting was blind to the experimental groups.
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