Slides were brought to room temperature and dehydrated in PBS. Following a 5-min post fixation with 4% PFA, antigen retrieval was performed as shown in Table 1. For unconjugated antibodies, slides were blocked with 1% goat serum for 1 h before incubating with primary antibodies at 4°C in a humified chamber. Slides were washed and incubated with secondary antibody for 45 min at room temperature. Sections were mounted with ProLong Gold Mountant with DAPI and imaged on a Leica DMI3000B microscope using a Leica DFC3000G camera or a Leica SP5 inverted confocal microscope with the latest Leica imaging suite. Images were reconstructed and pseudocolored on Fiji.
Dfc3000g camera
Manufactured by Leica
Sourced in Germany
The Leica DFC3000G camera is a high-performance digital camera designed for microscopy and laboratory applications. It features a CMOS sensor that captures images with a resolution of up to 5 megapixels. The camera provides a variety of connectivity options, including USB 3.0 and Ethernet interfaces, allowing for easy integration with a range of laboratory equipment and software.
Automatically generated - may contain errors
Lab products found in correlation
Dm il led microscope, by Leica (8 mentions)
Dmi3000b microscope, by Leica (4 mentions)
M165fc microscope, by Leica (4 mentions)
Dmi4000b microscope, by Leica (3 mentions)
Mdg41 microscope, by Leica (3 mentions)
Mz10f stereomicroscope, by Leica (3 mentions)
Application suite x software, by Leica (3 mentions)
Dm1000 model microscope, by Leica (3 mentions)
Sp5 inverted confocal microscope, by Leica (2 mentions)
Application suite 4, by Leica (2 mentions)
Mz10f microscope, by Leica (2 mentions)
Glass microscope slide, by Thermo Fisher Scientific (2 mentions)
Dmi4000b, by Leica (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Prism 9, by GraphPad (2 mentions)
Mzlfiii microscope, by Leica (2 mentions)
Fm4 64 dye, by Thermo Fisher Scientific (2 mentions)
Nanozoomer xr digital slide scanner, by Hamamatsu Photonics (2 mentions)
Tcs sp5 confocal microscope, by Leica (2 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
65 protocols using dfc3000g camera
1
Antigen Retrieval and Immunofluorescence Imaging
2
Keratin Peptide Penetration in Textured Hair
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Penetration studies were performed on relaxed textured hair. People with this hair type tend to use long‐duration treatments, including those designed for overnight use (while wearing a cap). To understand penetration behaviour, three‐hour soaking was selected for this study. A total of 10 mg of hair (~20–30 × 1‐inch hair fibres) was soaked in 1.5 mL labelled keratin peptide solution such that hair fibres were fully immersed in the solution. Hair samples were incubated for 3 h with gentle rocking. After incubation, hair was rinsed in DI water for 1 min and air‐dried on a paper towel. Hair fibres were embedded as a bundle in OCT compound (Optimal cutting temperature, Sakura Finetek USA, Inc, Torrance, CA, USA) prior to cryosectioning. Several hair cross‐sections (5 µm) were collected for image analysis.
Hair cross‐section images were collected on a Leica DM8 (Leica biosystems, Wetzlar, Germany), equipped with the Leica DFC3000 G camera. Images were acquired using the Leica Application suite X (LAS X) software. Each image was recorded using two separate channels: green (Excitation:460–500 nm/DC:505 nm/Emission:512–542 nm), exposure 150 ms, and red (Excitation:532–558 nm/DC:565 nm/Emission:570–640 nm), exposure 2 ms.
Hair cross‐section images were collected on a Leica DM8 (Leica biosystems, Wetzlar, Germany), equipped with the Leica DFC3000 G camera. Images were acquired using the Leica Application suite X (LAS X) software. Each image was recorded using two separate channels: green (Excitation:460–500 nm/DC:505 nm/Emission:512–542 nm), exposure 150 ms, and red (Excitation:532–558 nm/DC:565 nm/Emission:570–640 nm), exposure 2 ms.
3
Antigen Retrieval and Immunofluorescence Imaging
4
Soft Agar Colony Formation Assay
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GSC soft agar colony formation assays were performed as described (Gil-Ranedo et al, 2011 (link)). Briefly, 2 ml of complete DMEM/F12 medium with 0.35% agar were added into M6 wells and 1 × 104 (GSC-5) or 5 × 103 (GSC-8) puromycin-selected cells seeded on top in another 2 ml of complete DMEM/F12 medium with 0.5% agar. GSC-5 and GSC-8 cells were incubated for 9 and 10 days, respectively. Colony size and morphology were recorded with a Leica DM IL LED microscope coupled to a Leica DFC3000 G camera. For colony numbers, 1 ml per well of complete DMEM/F12 medium containing 600 µg of MTT (Sigma) was added overnight to allow scoring at lower magnification towards scoring. Imaging was conducted on a Leica DM1000 LED microscope coupled to a Leica MC170 HD camera. Colony number scored using nucleus counter plugin included in Fiji/ImageJ (Schindelin et al, 2012 (link)).
5
Magnetically Guided Bacterial Microswimmers
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Microtubes were added to bacteria in motility media and allowed to incubate at 37°C and 150 rpm for 30 min. The solutions containing the biohybrid swimmers were imaged in petri dishes with glass coverslip bottoms (Cellview Cell Culture Dish, Greiner Bio-One) at ambient temperature. Videos and images were acquired using bright field microscopy with an inverted Leica (Wetzlar, Germany) DMI3000B microscope, Leica DFC3000G camera, and Leica Application Suite v.4.5.0 software. For magnetically guided microswimmers, motion control experiments were conducted using an electromagnetic coil system. The system consisted of four orthogonally oriented iron-core electromagnets. The coils were built onto a custom-made microscope stage to surround a 35 mm Petri dish and was placed onto an inverted Zeiss (Carl Zeiss, Inc., Oberkochen, Germany) Axio Observer A1 microscope with a 20x objective (Figure 4 and Figure S1). The input current for the coils was controlled by motor drivers and an Arduino microcontroller board, and the ~8 mT magnetic field strength was calibrated using a Lake Shore Cryotronics (Darmstatdt, Germany) Model 460-3-Channel Gaussmeter.
Microswimmers were recorded with a Zeiss Axiocam 503 CCD camera.
Microswimmers were recorded with a Zeiss Axiocam 503 CCD camera.
6
Quantitative Immunofluorescence Imaging of Tumor Cells
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Tissue sections were cut at 5 µm and fixed with acetone for 10 min, dried, washed, and blocked with PBS/10% donkey serum/0.3% Triton for 30 min prior to immune staining. Next, sections were incubated with the primary antibody overnight at 4°C, washed and incubated with the donkey anti-rat IgG Alexa Fluor 488 for 2 h at room temperature in the dark. Sections were covered with Vectashield, and kept subsequently at room temperature for 2 h and at 4°C overnight. Sections were examined microscopically (Leica, DM IL, 200x magnification) and photographed (Leica DFC 3000G camera and LAS4 software). Recorded photographs were analyzed using Fiji software.31 (link) The number of pictures from each tumor ranged between 12 and 16, and using an in-house developed algorithm, the mean number of positively stained cells was determined, and normalized for percentages of nucleated cells (DAPI staining).
7
Annexin V Live Cell Labeling Protocol
8
Immunofluorescence Staining of IBA1 and OCT4
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Cells were fixed using paraformaldehyde (PFA) 4% for 20 min and permeabilized using Triton X-100 0.1% for 10 min. Unspecific binding was blocked by incubation with 5% BSA for 1 h at room temperature. Incubation with primary antibody against IBA1, OCT4, was conducted overnight at 4 °C. Secondary anti-rabbit antibodies coupled to Alexa Fluor 488 (Invitrogen, A11034) or 568, (Invitrogen, A10037) were incubated at room temperature. Nuclei were counterstained with DAPI. Images were acquired using a Leica DFC3000 G camera and acquired using Leica Las 4.3 software.
9
Immunocytochemistry for α-SMA Expression
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Immunocytochemistry (ICC) was performed as previously described [24 (link)]. Briefly, cells were seeded at a density of 5x104 cells/well onto sterile glass cover slips in 6-well plates. After overnight attachment, cells were treated with TGF-ß1 and/or drugs or control conditions. After 72 h, the cells were fixed using ice-cold methanol, washed in PBS, and blocked using 10% donkey serum in PBS for 2 h. This was followed by primary antibody incubation using an anti–α-SMA antibody (1:1000; Sigma-Aldrich, Gillingham, United Kingdom) for 2 h. Cover slips were washed three times using PBS and cells were incubated with secondary antibody in PBS (1:1000; Abcam, Cambridge, United Kingdom) for 2 h. After washing, coverslips were mounted using VECTASHIELD® Antifade Mounting Media with DAPI (Vector Laboratories, Newark, California, United States). Images were captured using a Olympus microscope with a Leica DFC3000 G camera.
10
Isolation of Adipose Mesothelial Cells
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Adipose associated mesothelial cells were isolated as described previously (Darimont et al., 2008 (link)). Epididymal adipose depots were harvested from 6 weeks-old male MuralChaser mice treated with doxycyline as described above. Intact whole adipose depots were placed in 10 mL of PBS containing 0.25% trypsin for 20 min at 37°C with continuous end over end rotation. Next adipose tissues were removed, and remaining solution containing isolated cells was centrifuged at 600 x g for 5 min. The media was aspirated, and the cell pellet was resuspended in growth media (10% FBS in DMEM/F12 (Invitrogen)) and plated in a 12-well collagen-coated plate. The cells were incubated at 37°C in 10 CO2 and the media was replaced daily. Images were obtained using a Leica DMIL LED microscope and a Leica DFC3000g camera.
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