To monitor and localize ROS production by live confocal imaging, mast cells (1.5 × 106 cells) were immobilized overnight in MatTek glass bottom microwell dishes (MatTek Co., Ashland, MA) using a biocompatible anchor for membrane (BAM; SUNBRIGHT® OE-040CS, NOF Corporation, Tokyo, Japan). To label granules and detect ROS, cells were incubated with a cocktail of probes (50 nM LysoTrackerTM Red DND-99 and 5 μM CellROXTM Deep Red) for 30 min. Cells were subsequently washed and kept in PBS. Images were immediately recorded at the indicated time intervals at 37 °C and 5% CO2 at baseline (initial 72 min), and after addition of mefloquine (for another 112 min) using a Nikon Ti2-E microscope, equipped with an X-LIGHT V2 L-FOV spinning disk with a pinhole size of 60 µm (Crest Optics). A 100X/1.45 NA oil objective (Nikon) and a Prime 95B 25 mm camera (Photometrics) were used to capture the images. To monitor cell death by live confocal imaging, BAM-anchored WT and serglycin−/− mast cells (1.5 × 106 cells) were incubated with a cocktail of Annexin V and DRAQ7 in a glass bottom 6-well plate (MatTek Co.). Thereafter, cells were treated with either PBS or mefloquine and images were immediately recorded at indicated time intervals, at 37 °C and 5% CO2, through a 40X/0.6 NA air objective on a Nikon Ti2-E microscope.
Ti2 e microscope
Manufactured by Nikon
Sourced in United States, Japan
The Ti2-E microscope is a high-performance inverted microscope designed for a variety of laboratory applications. It features a stable and rigid frame, providing a stable platform for precise imaging and analysis. The Ti2-E is equipped with advanced optics and illumination systems, enabling clear and detailed observation of samples.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (3 mentions)
Bsi scmos camera, by Teledyne (2 mentions)
Csu w1 spinning disk unit, by Yokogawa (2 mentions)
Fm4 64, by Thermo Fisher Scientific (2 mentions)
Metamorph software, by Molecular Devices (2 mentions)
Plan apo 100x 1.4na phase contrast oil objective, by Nikon (2 mentions)
Wright s blood stain, by Thermo Fisher Scientific (2 mentions)
Nis elements software, by Nikon (2 mentions)
Glass bottom 6 well plate, by MatTek (1 mentions)
Sunbright oe 040cs, by NOF corporation (1 mentions)
Glass bottom microwell dishes, by MatTek (1 mentions)
Ula plate, by Corning (1 mentions)
Draq5, by Thermo Fisher Scientific (1 mentions)
Nis elements viewer, by Nikon (1 mentions)
Vectashield antifade mounting media, by Vector Laboratories (1 mentions)
Supermount, by BioGenex (1 mentions)
Nis elements br 3, by Nikon (1 mentions)
Paraformaldehyde (pfa), by Solarbio (1 mentions)
80i microscope, by Nikon (1 mentions)
50 protocols using ti2 e microscope
1
Monitoring mast cell ROS and death
2
Sphere Formation Assay for Ovarian Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sphere formation of OV90, OVCAR8 and OVCAR3 cells was performed as previously described (15 (link)). Cells were seeded at 2,000 cells/well in 96-well ULA plates (3474, Corning, Corning, NY), in TEM with indicated drugs for 7 days, fresh culture medium containing growth factors and the drugs was replenished every 48 h. After 7 days the spheres were incubated with DRAQ5 (Thermo Fisher Scientific, Waltham, MA, USA) at 1 µM for 15 min prior to imaging using an inverted Nikon Ti2-E microscope (Nikon, Melville, NY), equipped with a Yokogawa SoRa CSU-W1 spinning disk unit (Yokogawa, Sugar Land, TX) and a BSI sCMOS camera (Teledyne Photometrics, Tuscon, AZ). Images of spheroids were acquired using the automated acquisition module (JOBS) to image each well using a 10× plan-apochromat N.A. 0.45 objective lens, 200 ms exposure. Quantification of spheroids was performed using NIS Elements software version 5.30 (Nikon, Melville, NY). Images were processed using Segment.ai trained on human-recognized, hand-traced spheroids. Segment.ai learned how to trace spheroids in subsequent images. The number of spheroids measuring an area of >1,000 μm2 were counted.
3
Immunofluorescence Microscopy Procedure
4
Immunofluorescence Analysis of p65 and YAP
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BMDMs were placed in 6-well plates or cell slides. BMDMs were treatment with LPS with or without XMU-MP-1 and fixed with paraformaldehyde (Solarbio).
BMDMs in 6-well plates were incubated with anti-p65 antibody (1:500, 8242, Cell Signaling Technology) overnight at 4 °C. As secondary antibodies, Alexa 488 (Cell Signaling Technology) were added in well plates for 1h at room temperature, and then added Hoechst (TIANGEN) for 15 min in dark. These 6-well plates were visualized with a Nikon Ti2-E microscope (Nikon). Images were analyzed by Nikon NIS-Elements Br 3.0 software (Nikon). To quantify the nuclear translocation of p65, ten files from each group were analyzed for the p65 high positive in nuclei (%). The p65 high positive in the nuclei (%) was calculated (p65 high positive in the nuclei (%) = the number of p65 high positive in the nuclei ×100 / the number of total nuclei) 14 (link).
BMDMs on cell slides were incubated with anti-p65(1:500, 8242, Cell Signaling Technology) and anti-YAP (1:50, sc-101199, Santa Cruz) antibodies. As secondary antibodies, Alexa 594 and 488 (Cell Signaling Technology) were added for 1h at room temperature in dark, and then sections were sealed by fluorescent mounting medium with DAPI (ZSGB-BIO) in dark. These sections were visualized with a Nikon 80i microscope (Nikon). Images were analyzed with Nikon NIS-Elements Br 3.0 software (Nikon).
BMDMs in 6-well plates were incubated with anti-p65 antibody (1:500, 8242, Cell Signaling Technology) overnight at 4 °C. As secondary antibodies, Alexa 488 (Cell Signaling Technology) were added in well plates for 1h at room temperature, and then added Hoechst (TIANGEN) for 15 min in dark. These 6-well plates were visualized with a Nikon Ti2-E microscope (Nikon). Images were analyzed by Nikon NIS-Elements Br 3.0 software (Nikon). To quantify the nuclear translocation of p65, ten files from each group were analyzed for the p65 high positive in nuclei (%). The p65 high positive in the nuclei (%) was calculated (p65 high positive in the nuclei (%) = the number of p65 high positive in the nuclei ×100 / the number of total nuclei) 14 (link).
BMDMs on cell slides were incubated with anti-p65(1:500, 8242, Cell Signaling Technology) and anti-YAP (1:50, sc-101199, Santa Cruz) antibodies. As secondary antibodies, Alexa 594 and 488 (Cell Signaling Technology) were added for 1h at room temperature in dark, and then sections were sealed by fluorescent mounting medium with DAPI (ZSGB-BIO) in dark. These sections were visualized with a Nikon 80i microscope (Nikon). Images were analyzed with Nikon NIS-Elements Br 3.0 software (Nikon).
5
Co-Detection of Insulin Receptor and Keratin 5
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Insr and Krt5 were co-detected using the RNAscope 2.5 HD Duplex Assay and RNA-Protein Co-detection Ancillary Kit (Advanced Cell Diagnostics, Hayward, CA, USA). Deparaffinized and dehydrated slides were pretreated using the RNAscope Hydrogen Peroxide (10-min), and RNAscope Co-Detection Target Retrieval (20-min) reagents. Basal cells were labeled with a keratin 5 antibody overnight then slides were fixed in 10% formalin (30 min). Slides were treated with Protease Plus reagent (30-min), hybridized with Insr-C2 which recognizes exons 3–5 of the Insr gene, Polr2A-C2 (positive control), or DapB-C2 (negative control) probes (2 h, Advanced Cell Diagnostics), amplified using AMPS 1–6, and C2 signal was visualized using the Detect Red reagents (10 min). Following treatment with Co-Detection Blocker (30 min), a goat anti-rabbit-HRP secondary antibody was applied (1 h, Cell Signaling) and keratin 5 was visualized using 3,3-diaminobenzidine tetrahydrochloride (10 min). Slides were counterstained in 50% Gill’s Hematoxylin I (Thermo Fisher Scientific, Waltham, MA, USA). Cover slips were applied with VectaMount (Vector Laboratories, Inc., Burlingame, CA, USA). Images were captured using a Nikon Ti2-E microscope and DS-RI2 camera (Nikon Instruments Inc., Melville, NY, USA).
6
Cyclic Immunofluorescence Imaging of FFPE TMA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For GIT dataset, formalin-fixed paraffin-embedded (FFPE) tissue microarray (TMA) slides were obtained from Pantomics (Pantomics, DID381) Tissue TMA samples for Supplementary Figures 12 , 13 , and 14 were obtained from Department of Pathology at University of Pittsburgh Medical Center Presbyterian Hospital. The slides went through cyclic immunofluorescence antigen retrieval protocol10 (link). The corresponding figure slides were stained in cycles with 1:200 dilution of Anti-Sodium Potassium ATPase antibody (Abcam ab198367, clone EP1845Y), 1:100 dilution of P53 antibody (Abcam ab270192, clone SP5), 1:50 dilution of EZH2 antibody (CST 45638, clone D2C9), and 1:100 dilution of LAMIN A/C antibody (CST 8617, clone 4C11) overnight at 4°C in the dark, followed by staining with Hoechst 33342 (CST 4082S) for 10 minutes at room temperature in the dark. TMA images were acquired using a 0.95 NA and a 40X objective on a Nikon Ti2E microscope.
Seventy five, 1000 × 1000 high quality regions were identified and extracted from the TMA images and saved as tiff images. Expert pathologists independently annotated these images. The annotations were done using Cellthon, a python based cell annotation graphical user interface (GUI) we created using Tkinter toolkit55 . Together these 75 images and their cell and nucleus annotations comprise the GIT dataset.
Seventy five, 1000 × 1000 high quality regions were identified and extracted from the TMA images and saved as tiff images. Expert pathologists independently annotated these images. The annotations were done using Cellthon, a python based cell annotation graphical user interface (GUI) we created using Tkinter toolkit55 . Together these 75 images and their cell and nucleus annotations comprise the GIT dataset.
7
Fluorescence Microscopy of Cellular Membranes
8
Spatial Transcriptomic Analysis of Amyloid-Beta
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fresh frozen tissue samples were sectioned on a HM525NX cryostat (Fisher) at −15°C for 10μm thick sections that are immediately mounted onto 10x Genomics Visium slides. Slides were individually stored in slide mailers (sealed airtight in a plastic bag) at −80°C until staining. We followed 10x Genomics Methanol Fixation, Immunofluorescence Staining & Imaging for Visium Spatial Protocols (Rev C), except after tissue sections were fixed in methanol and blocked, the sections were incubated with Amylo-glo (1:100; TR-300, Biosensis) for 20 min. Sections were then incubated with the primary antibody OC (1:500 for mouse, 1:200 for human; polyclonal, AB2286, Millipore) and respective secondary antibody (1:400; goat anti-rabbit secondary antibody Alexa Fluor 488, Life Tech or Alexa Fluor 647, Life Tech). Immediately after immunostaining, capture areas were imaged on a widefield Nikon Ti2-E microscope at 20X magnification. Spatial transcriptomic libraries were then generated from the tissue sections according to the 10x Genomics Visium User Guide (Rev E). Library quantification, quality check, and sequencing were performed as previously described, but sequencing depth was based on an estimated 60% tissue area coverage per sample for 50,000 read pairs per covered spot.
9
Time-Lapse Imaging of TGF-β-Induced Cellular Changes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Time-lapse images were taken with a Nikon Ti2-E microscope with differential interference contrast (DIC) and TRITC channels (Excitation wavelength is 555 nm and Emission wavelength is 587) (20× objective, N.A. = 0.75). The cell culture condition was maintained with Tokai Hit Microscope Stage Top Incubator. For the 4 ng/ml TGF-β (R&D Systems 240-B) experiment, cells were imaged every 5 min with the DIC channel and every 10 min with the TRITC channel for 2 days. The exposure time for DIC was 100 ms and the exposure time for the TRITC channel was 30 ms. For the 1 ng/ml TGF-β experiment, cells were imaged every 5 min with the DIC channel and every 15 min with the TRITC channel for 3 days. The exposure time for DIC was 100ms and the exposure time for the TRITC channel was 30ms. While taking the images, all the imaging fields were chosen randomly.
10
Quantitative Analysis of Calcification in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed with 4% PFA on the calcification matched wells for 10 min and then washed with 1x PBS twice. 1:1000 DAPI in 0.01% Triton X-100 in 1x PBS was placed on the cells overnight at 4 °C. After incubation, cells were washed in 1x PBS twice and imaged with high content analysis Nikon Ti2-E microscope using a high-speed PCOS camera. Images obtained were analyzed with high content analysis software with automated cell counting, cell growth analysis, proximity analysis, and Nikon propriety JOBS analysis. Calcium content (µg) was normalized to cell number. Each experimental replicate is representative of 3 experimental repeats for each group.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!