Sections of tumor xenografts from the dLAN, dLAN + PTX, dLAN + MLT, and dLAN + MLT + PTX treatment groups were Formalin-fixed and paraffin-embedded into tissue blocks. Tissue sections were cut to 4-μM thickness, mounted on polylysine-coated slides, de-waxed, rehydrated in ethanol, and rinsed in phosphate-buffered saline. Antigen retrieval was performed by pretreating slides with citrate at pH 6 with microwaving for 10 minutes, and endogenous peroxidase activity was blocked using 0.3% H2O2. The slides were incubated with anti-pSTAT3 antibody (15μg/ml) and biotin-labeled secondary antibody, streptavidin/peroxidase, diminobenzidine, and counter stained with Hematoxylin/Eosin. Sections were imaged at a magnification of X 100, using an Olympus 1X70 microscope with a digital camera (Olympus, Tokyo, Japan).
1x70 microscope
Manufactured by Olympus
Sourced in Japan
The Olympus 1X70 microscope is a high-performance optical instrument designed for laboratory use. It features a sturdy and ergonomic design, providing a stable platform for precise observations. The microscope is equipped with advanced optics that deliver clear, detailed images, making it suitable for a variety of scientific applications.
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Lab products found in correlation
Delta vision system, by Olympus (2 mentions)
Sc 35 camera, by Olympus (2 mentions)
Deconvolution system, by Olympus (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Mouse anti flag, by Merck Group (1 mentions)
Acridine orange, by Merck Group (1 mentions)
Cxp software, by Beckman Coulter (1 mentions)
Fc500 flow cytometer, by Beckman Coulter (1 mentions)
Avance 3 700 spectrometer, by Bruker (1 mentions)
Tlc silica gel 60 rp 18 f254s, by Merck Group (1 mentions)
Hp 8453, by Hewlett-Packard (1 mentions)
Jms 700 mstation mass spectrometer, by JEOL (1 mentions)
Sephadex lh 20, by Merck Group (1 mentions)
Tlc silica gel 60 f254, by Merck Group (1 mentions)
Isolera one, by Biotage (1 mentions)
Alexa fluor 488 antibody, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Softworx 3, by Cytiva (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
12 protocols using 1x70 microscope
1
Immunohistochemical Analysis of Tumor Xenografts
2
Immunostaining Protocol for Germ Line Analysis
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FISH and immunostaining were performed as in Silva et al (2014) [62 (link)]. Briefly, immunostaining was performed as follows: germ lines from 18–24 hours post L4 adults were dissected in EGG buffer (118 mM NaCl, 48 mM KCl2, 2mM CaCl2, 2mM MgCl2, 5mM HEPES) containing 0.1% Tween and immediately fixed in 1% paraformaldehyde for 5 minutes. Slides were frozen in liquid nitrogen, then immersed for at least 1 minute in methanol at –20°C and transferred to PBST (1x PBS, 0.1% Tween). Blocking in 1% BSA in PBST was carried out for 1 hour. Primary antibodies were incubated overnight at room temperature, slides were then washed 3 times for 10 minutes in PBST and secondary antibodies were added and incubated for 2 hours at room temperature. Following 2 washes in PBST, the slides were counterstained with DAPI and mounted using Vectashield. All images were acquired as three-dimensional stacks on a Delta Vision system equipped with an Olympus 1X70 microscope. The following primary antibodies were used: rabbit anti-HTP-1, 1:400, chicken anti-SYP-1, 1:350, rabbit anti-RAD-51, 1:10.000 (SDIX), mouse anti-FLAG, 1:500 (Sigma). Secondary antibodies were used at 1:500 dilution and included: goat anti-rabbit Alexa488-conjugated, goat anti-chicken Alexa555-conjugated and goat anti-mouse Alexa488-conjugated (Life Technologies).
3
Acridine Orange Staining for Cell Imaging
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Acridine orange (Sigma-Aldrich, A6014, St. Louis, MO, USA) staining solution was prepared in PBS at a ratio of (1:10,000) to reach to a final concentration of 100 ng/mL (prepared in the dark), added to cells, and incubated for 15 min at 37 °C. Plates were washed again with 1X PBS and fresh medium was added. Images were taken using an Olympus 1X 70 microscope and an Olympus SC 35 camera, as performed in previous publications [32 (link),33 (link)].
4
Quantifying Oocyte Chromosome Morphology
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Images of DAPI-stained -1 and -2 diakinesis oocytes were acquired using a Delta Vision system equipped with an Olympus 1X70 microscope. Deconvolved image stacks were either used directly for visual counting, or were converted into maximum intensity projections, cropped to the size of an individual nucleus using ImageJ and analysed in CellProfiler to define the boundaries of each DAPI-stained body and to calculate its area in pixels. Following analysis of oocytes from control genotypes (Figure 5D ), the following areas were used to classify DAPI-stained bodies: bivalents 500–1000 pixels, univalents 250–500 pixels, sister chromatids 120–250 pixels. DAPI-stained bodies with an area smaller than 120 pixels were counted as chromosome fragments.
5
Acridine Orange Cell Viability Assay
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200,000 cells were seeded in 6-well plates and permitted to adhere overnight and drug-treated the next day. At various time points, drug was removed and cells washed once with 1X PBS. Acridine orange dye was diluted in PBS in a ratio of 1:10000 (prepared in the dark) and added to cells for staining, and incubated for 15 min. Dye then was aspired and plates were washed with 1X PBS and fresh medium was added. Photographs were taken with an Olympus 1X 70 microscope and an Olympus SC 35 camera.
Flow analysis was utilized to count the cell population positively stained with acridine orange. Treated cells were harvested, collected, and centrifuged at speed of 1500 rpm. Supernatant was removed and pellets were resuspended in 1 mL of fresh medium. Cell suspension was filtered through standard flow cytometry 40 µm filter (BD falcon). A 10:1000 dilution of acridine orange in 1X PBS was prepared (in the dark) and protected from light until ready for use. At flow cytometry, 10 μL of acridine orange solution was added to per sample to make the dilution of 1:10000. When analyzed by flow cytometry using the FC500 Flow cytometer with CXP software (Beckman Coulter, USA), the acridine orange dye is excited at wavelength 525 nM for green fluorescence and 620 nM for red fluorescence.
Flow analysis was utilized to count the cell population positively stained with acridine orange. Treated cells were harvested, collected, and centrifuged at speed of 1500 rpm. Supernatant was removed and pellets were resuspended in 1 mL of fresh medium. Cell suspension was filtered through standard flow cytometry 40 µm filter (BD falcon). A 10:1000 dilution of acridine orange in 1X PBS was prepared (in the dark) and protected from light until ready for use. At flow cytometry, 10 μL of acridine orange solution was added to per sample to make the dilution of 1:10000. When analyzed by flow cytometry using the FC500 Flow cytometer with CXP software (Beckman Coulter, USA), the acridine orange dye is excited at wavelength 525 nM for green fluorescence and 620 nM for red fluorescence.
6
Characterization of Organic Compounds
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The melting point was taken using an Electrothermal 9300 (Electrothermal Engineering LTD). UV spectra were obtained from a Hewlett-Packard HP8453 diode array spectrometer. NMR experiments were carried out with a Bruker AVANCE III 700 spectrometer (Ettlingen, Germany). Chemical shifts (δ) are reported in parts per million (ppm), referencing the solvent used. ESIMS data were obtained on a Waters Acquity Ultra Performance LC-MS system LCA 048 (Milford, MA, USA). LRFABMS and HRFABMS spectra were obtained on a JMS-700 M Station Mass Spectrometer (JEOL Ltd., Tokyo, Japan). Column chromatography and MPLC were performed on Sephadex LH-20 (25–100 μm, Sigma-Aldrich, St. Louis, MO, USA) and Biotage Isolera One equipped with Biotage® SNAP ULTRA C18 Cartridges (Uppsala, Sweden), respectively. Thin-layer chromatography was conducted on TLC Silica gel 60 F254 and TLC Silica gel 60 RP-18 F254S (Merck, Darmstadt, Germany). Fluorescence microscopy was carried out with an Olympus 1X70 microscope (Japan). Image J (NIH, Bethesda, MD, USA) and Focus Lite (Focus Co., Seoul, Republic of Korea) were used for image analysis.
7
Recombinant Protein Expression and Immunofluorescence Assay
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HEK293T cells were grown in 24-well plates coated with poly-L-lysine (Sigma-Aldrich, St. Louis, MO, USA). Cells were transfected with each recombinant plasmid (500 ng) in optimized minimal essential medium (MEM) (Opti-MEM; Gibco) using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) at a 1:2 ratio, and incubated for 24 h at 37 °C. MDCK cells were also infected with rescued plaque purified viruses and incubated at 37 °C for 2 days. Following incubation, medium was removed and the cells were fixed with 4% paraformaldehyde in PBS for an hour at room temperature (RT). After the fixation process, the cells were washed with PBS, blocked with 4% BSA in PBS, and incubated with primary anti-HA stalk murine mAb KB2, or anti-gH CMV specific mAb 10C10, at a dilution of 1:500 for 2 h at 37 °C. Cells were washed five times with PBS and incubated at RT for 1 h with an anti-mouse Alexa Fluor-488 antibody (Life Technologies, Carlsbad, CA, USA) diluted 1:1000 in 1% BSA–PBS. Fluorescent signals were visualized and captured by using an Olympus 1X-70 microscope.
8
Quantitative Analysis of Chromatin in Oocyte Nuclei
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Worms of indicated genotypes and treatments were dissected and processed for immunostaining as described in the main methods, including the final DAPI staining step. Images were acquired as 3D stacks using a ×100 lens in a Delta Vision Deconvolution system equipped with an Olympus 1X70 microscope. Images were deconvolved using SoftWoRx 3.0 (Applied Precision) and mounted in Photoshop. The number and appearance of DAPI-stained bodies in diakinesis and metaphase I oocytes were scored in 2D projections of three-dimensional intact germlines/embryos. Projections of diakinesis and metaphase I oocytes result in some overlap of DAPI-stained bodies, especially in situations where loss of SCC results in high numbers of DAPI-stained bodies; therefore in some cases, the number of DAPI-stained bodies may represent a slight underestimation of the overall number of individual chromatin bodies present.
The area in pixels of DAPI-stained bodies in diakinesis oocytes (Figure 4A ) was calculated by generating maximum intensity projections of diakinesis nuclei and using CellProfiler to obtain the size in pixels of individual DAPI-stained bodies identified in 2D projections.
The area in pixels of DAPI-stained bodies in diakinesis oocytes (
9
Quantitative Lung Morphometry Analysis
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H&E stained sections were photographed using an Olympus microscope with an in built digital camera system at 100× magnification (Olympus, Tokyo, Japan). Alveolar size was estimated from the mean chord length of the airspace, as previously described [19 (link)]. Alveolar septal wall thickness was estimated using Image J software, adapting the method described previously for bone trabecular thickness, for the lung [19 (link)]. 10× magnification images were obtained with Olympus 1X70 Microscope using Cellsens Dimension software. Radial alveolar count (RAC) and secondary septal crests were done as previously described [24 (link),25 (link)].
10
Imaging Meiotic Divisions in C. elegans Embryos
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24 hrs post L4 hermaphrodites were dissected to release embryos in a drop of 60% v/v Leibowitz-15 media, 20% fetal bovine serum, 25 mM HEPES pH 7.4, 5 mg/ml inulin. Embryos were then mounted on 2% agarose pads and imaged with a Delta Vision system equipped with an Olympus 1X70 microscope. Images of the meiotic divisions were acquired as series of 1 µm-spaced Z stacks (9–12 section) with a regular time lapse of 5 s intervals. Videos of these time series were created using SoftWoRx.
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