After fixation with PFA, spleens were frozen-embedded in optimal cutting temperature (O.C.T.) compound (Tissue-Tek, VWR). 10 μm thick sections were obtained with a cryostat microtome (Microm HM 505e) and transferred to charged microscope slides (VWR). Slides were stored at –80° C, then thawed in PBS at room temperature, washed 3x in PBS, and stained with Hoechst (1:10,000 dilution in PBS) for 5 minutes. Slides were then washed 3x with PBS and coverslips were mounted with ProLong Gold (Life Technologies). One section was imaged for each tissue. Tissues were imaged with a Zeiss Axio Observer 7 (Zeiss) inverted fluorescent microscope with an Apotome.2 (Zeiss) with the 63x oil immersion objective. Images of the sections were captured with an Axiocam 702 mono camera (Zeiss).
Axiocam 702 mono camera
Manufactured by Zeiss
Sourced in Germany
The Axiocam 702 mono is a scientific-grade digital camera designed for microscopy applications. It features a monochrome sensor optimized for high-sensitivity image capture. The camera offers a resolution of 2.3 megapixels and supports a variety of output formats. It is intended for use with Zeiss microscope systems and software.
Automatically generated - may contain errors
Lab products found in correlation
Calcein am, by Biotium (2 mentions)
Ethidium homodimer 1, by Biotium (2 mentions)
Hoechst 33342, by Biotium (2 mentions)
Calibri 7 led light source, by Zeiss (2 mentions)
Mitosox, by Thermo Fisher Scientific (2 mentions)
Rhod 2 am, by Thermo Fisher Scientific (2 mentions)
Axio observer 7 microscope, by Zeiss (2 mentions)
Charged microscope slides, by Avantor (1 mentions)
Apotome 2, by Zeiss (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Axio observer 7, by Zeiss (1 mentions)
Mitoview green, by Biotium (1 mentions)
Lsm 700 laser, by Zeiss (1 mentions)
Zen 2.6 blue edition software, by Zeiss (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
Sc 514414, by Santa Cruz Biotechnology (1 mentions)
Tetramethylrhodamine methyl ester tmrm, by Biotium (1 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
Axiovert 200, by Zeiss (1 mentions)
8 protocols using axiocam 702 mono camera
1
Cryosectioning and Immunofluorescence of Spleen
2
Rapamycin Inhibits Plasmodium Schizont Invasion
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Synchronous ring-stage FRM1-HA parasites received either DMSO as the mock control (0.05%) or Rapamycin (Rap, 100 nM) and were incubated for approximately 48 h until they reached schizont stage. Here, they were mounted in an environmental chamber where they were filmed at 4 s per frame as using the AxioCam 702 Mono camera on a Zeiss Cell observer widefield microscope32 (link),33 (link). Video files were manipulated and labelled in FIJI and invasion data was graphed in GraphPad Prism.
3
Multimodal Imaging of Mitochondrial Function
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MitoView Green, TMRM, Calcein-AM, ethidium homodimer-1, and Hoechst 33342 were purchased from Biotium. MitoSox was purchased from Life Technologies. MPTP imaging was described previously [19 (link)]. Dye based calcium imaging was done with Rhod-2AM (Invitrogen, R1245MP) as per manufacturer’s protocol (including the production of dihyrdorhod-2 AM). Immunofluorescence with HMBG1 (CST # 3935), Bnip3 [CST # 3769 and Ab-196706 (Alexa Fluor 647)], 14-3-3β [sc-25276 (Alexa Fluor 488)], and Opa1 [sc-393296 (Alexa Fluor 488)] antibodies were performed in conjunction with fluorescent secondary antibodies conjugated to Alexa Fluor 466 or 647 (Jackson), when primary antibodies were not conjugated to a fluorophore. All epifluorescent imaging experiments were done on a Zeiss Axiovert 200 inverted microscope fitted with a Calibri 7 LED Light Source (Zeiss) and Axiocam 702 mono camera (Zeiss) in combination with Zen 2.3 Pro imaging software. Confocal imaging was done on a Zeiss LSM700 Spectral Confocal Microscope in combination with Zen Black, which was also used for colocalization analysis, while FRET imaging was done using a Cytation 5 Cell Imaging Multi-Mode Reader. Quantification, scale bars, and processing including background subtraction, was done on Fiji (ImageJ) software. Quantification of mitochondrial morphology was performed as previously described [43 ].
4
Confocal Microscopy Imaging Protocol
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Fluorescence microscopy was performed with the LSM 700 laser scanning confocal microscope (ZEISS), equipped with Axiocam 702 mono camera (ZEISS) and the images were captured at 10X magnification. A minimum of five brain sections from each animal were assessed for analysis. Confocal microscopy images were captured with the same microscope in confocal mode (software: Zen2.6 Black edition, ZEISS) and at 63X magnification oil immersion objective (N.A. 1.4) at 1024 × 1024 pixel resolution. The images were collected at a depth mid-way into the specimen, and the brightest focal plane with sharp morphological features was selected using the live acquisition feature and a 1 AU pinhole that enables a high-resolution image to be collected. All the quantitative analysis were on area of the 2D surface, and in cases wherein normalization was needed, we used a 2D measurement (e.g. imageframe area in mm2). Sample identity was blinded for all analyses and images were processed with Zen2.6 Blue edition software (ZEISS).
5
Immunofluorescence Analysis of Cell Markers
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Cells were grown in ibidi chamber slides (Gräfelfing, Germany), fixed, permeabilized, blocked with 10% serum and incubated overnight with primary anti-human antibodies: rabbit Claudin-7 (1:100, Thermo Fisher Scientific, Dreieich, Germany), rabbit E-Cadherin (1:100, Santa Cruz Biotechnology, Heidelberg, Germany), followed by a secondary AF488® goat anti-rabbit antibody (1:1000, 1 h, Molecular Probes, Darmstadt, Germany). Directly labeled antibodies were used: PE mouse CD142 antibody (1:50), AF488® mouse CD326 (EpCAM) Antibody (1:100, BioLegend), AF594® N-Cadherin (1:100, Wiesbaden, Germany), AF488® anti-p27/Kip1 Antibody (1:50, Novus Biologicals), AF594® mouse p53 Antibody (1:250, BioLegend), AF488® rabbit p21 Waf1/Cip1 Antibody (1:300, Cell Signaling Technology, Frankfurt am Main, Germany), AF546® mouse p16 Antibody (1:50, Santa Cruz Biotechnology). Nuclei were counterstained with DAPI. Cells were analyzed on a Zeiss Axio Observer 7 Microscope using a Axiocam 702 mono camera and the ZEN pro software.
6
Immunofluorescent Localization of ASC Protein
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The cells were fixed with a methanol–acetone mixture (500 µL of 1:1 methanol–acetone mixture was added to each well for 5 min at 4 °C) and were permeabilized with Triton (500 µL of 0.1% Triton in PBS were added to each well for 10 min at room temperature). After rinsing the coverslips with PBS, the cells were incubated with 4% BSA in PBS for 1 h at room temperature. The coverslips were incubated with the antibody against ASC (sc-514414, Santa Cruz Biotechnology, 150 µL, 1:500) overnight at 4 °C. After 24 h, the coverslips were rinsed again with PBS and were incubated with the secondary antibody for 1 h in the dark (AF555 anti-mouse, 1:200, 150 µL/well). The coverslips were rinsed with PBS, stained with DAPI and mounted on microscope slides. The staining was evaluated using an Axio Observer Z1 microscope with an Axiocam 702 mono camera (Zeiss, Oberkochen, Germany), and the stained area per cell was measured using ImageJ.
7
Live Cell Imaging and Quantification
8
Fluorescent Imaging of Cellular Stress Pathways
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H9c2 or PVNCs were cultured and treated with DOX for 24 hours, after which they were incubated with the appropriate dye for 30 minutes, washed with PBS three times, and then imaged in culture media. All imaging was done using a Zeiss Axiovert 200 inverted microscope equipped with a Calibri 7 LED Light Source (Zeiss) and Axiocam 702 mono camera (Zeiss). Calcein-AM, ethidium homodimer-1, tetramethylrhodamine methyl ester (TMRM), and Hoechst 33342 dyes were purchased from Biotium. MitoSOX was purchased from Life technologies while LysoTrackerRed DND-99, Rhod-2AM, and dichlorodihydrofluorescein diacetate (H 2 -DCFDA; DCF) were purchased from Invitrogen. Mitochondrial permeability transition pore imaging (mPTP) imaging was carried out with Calcein-AM and cobalt chloride using the same method previously described by our lab (28, (link)30, (link)31) (link). Mito-pHred, a plasmid-based biosensor (33) (link) and dihydro-Rhod2 (28) imaging were described previously.
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