HeLa and HeLa/SN100 cells were grown on Matek 2.5 cm plates with 1.5 glass coverslips until 80% confluent. Growth media was aspirated off and the cells were washed with PBS. Cells were then fixed with 1 ml of 10% formalin for 20 min at room temperature. Fixed cells were washed three times with PBS and then blocked for 1 h in 1 ml of PBS containing 5% BSA and 0.3% triton X-100. Cells were then incubated with primary antibodies (1:250) anti-Keratin 8 and (1:800) anti-Keratin 18 overnight at 4 °C. Cells were washed three times with PBS before incubation with the secondary antibodies, Alexa Fluor 568 goat anti-rabbit and Alexa Fluor 488 goat anti-mouse (Life Technologies) at 1:1,000 dilutions for 1 h at room temperature. Cells were then washed three times with PBS before imaging using a Nikon TiE inverted widefield fluorescence microscope. Image analysis was performed using ImageJ.
Ti e inverted widefield fluorescence microscope
Manufactured by Nikon
The Ti-E inverted widefield fluorescence microscope is a high-performance lab equipment designed for advanced fluorescence imaging. It features a stable and precise optical system, providing consistent and reliable results. The microscope is capable of capturing widefield fluorescence images, making it a versatile tool for various scientific applications.
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Lab products found in correlation
Ab138698, by Abcam (2 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Perfect focus system, by Nikon (1 mentions)
μ slide 8 well, by Ibidi (1 mentions)
Mab17504, by Abcam (1 mentions)
Cfi apo tirf 100x 1.49 objective, by Hamamatsu Photonics (1 mentions)
C11440 orca flash 4, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
A31556, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 donkey anti mouse, by Thermo Fisher Scientific (1 mentions)
Planapo 40 0.95 objective, by Nikon (1 mentions)
μ slide, by Ibidi (1 mentions)
8 protocols using ti e inverted widefield fluorescence microscope
1
Immunofluorescent Staining of Keratins
2
Intron and lncRNA Visualization in A375 Cells
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A set of probes, each 20 nt long, was designed specifically to the intron 10 and 15 of SND1 (32 probes) and intron 5 of SMYD3 (48 probes). The region of probe binding was selected to overlap the MACS peak regions identified in the ChIRP analysis while avoiding any repetitive sequences. Another set of 25 probes was designed to bind SPRIGHTLY lncRNA. The probes were ordered from LGC Biosearch LLC with a 3′-amino modification and were coupled with either tetramethylrhodamine for the intronic probes or Texas Red for the SPRIGHTLY probes. The A375 cells (both wild type and the cell line with SPRIGHTLY knockout) were cultured on glass coverslips, fixed, permeabilized, and hybridized with lncRNA probe and one of the intron probe sets. The next day, the unbound probes were washed off, and the coverslips were mounted and imaged using a Nikon Ti-E inverted wide-field fluorescence microscope with a 100× objective and a cooled charge-coupled device pixis camera.
3
Immunofluorescence Analysis of Differentiating Cells
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Immunofluorescence staining was performed on differentiating cell cultures at five different time points (D0, D7, D21, D30, and D40). Cells were washed with PBS and fixed in 4% formalin solution for 15 min at room temperature followed by a wash in PBS containing 0.005% Triton X-100 (PBS-T). Cells were permeabilized by treatment with PBS containing 0.5% Triton X-100 for 10 min, washed in PBS-T, and incubated with primary antibodies and 500 nM DAPI overnight. The next day, the cells were washed three times with PBS-T and incubated with fluorescent-conjugated Alexa secondary antibodies for 2 h at room temperature, followed by a PBS-T wash. The primary antibodies used in this study were anti-PAX3 mouse monoclonal antibody (1:200 dilution, DSHB), anti-PAX7 mouse monoclonal antibody (1:200 dilution, DSHB), anti-Myogenin mouse monoclonal antibody (1:250 dilution, DSHB), anti-Titin mouse monoclonal antibody (1:200 dilution, DSHB), and anti-DUX4 E5-5 rabbit polyclonal antibody (1:1000 dilution, Abcam). Secondary antibodies included Alexa 448, 594 and 647 anti-rabbit or anti mouse at 1:5000 dilution (Life Technologies). Images were captured with a Nikon TiE inverted widefield fluorescence microscope and processed using the elements software at the University of Washington’s Lynn and Mike Garvey Cell Imaging Laboratory.
4
High-Throughput Microscopy of Bacterial Strains
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All imaging was performed on a Nikon Ti-E inverted wide-field fluorescence microscope with an encoded XY-stage and the Nikon Perfect Focus System. Image capture was performed using an Andor Neo sCMOS camera, selected for its large field of view (2560 × 2160 pixels) and high sensitivity, and a 60X Plan-Apo oil-immersion objective (1.4 NA). The calibrated pixel size was 108 nm, meeting the Nyquist Criterion of two pixels per diffraction-limited spot. Fluorescence excitation was generated by a high-intensity mercury lamp. Image acquisition was controlled by NIS-Elements. Due to finite exposure time, duration of stage translocation between samples and the autofocus time, 48 strains could be imaged at 6–8 min intervals.
5
Immunofluorescence Imaging of T. gondii
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HFFs were grown to confluence in an 8-well μ-slide (Ibidi) and infected with T. gondii strains for 24 h. The slides were fixed with 4% w/v formaldehyde (Sigma) in phosphate-buffered saline (PBS) (Sigma). The cells were permeabilised with 0.2% v/v Triton X-100 (Sigma) for 15 min or 1 min and blocked with 2% w/v bovine serum albumin (Sigma) for 1 h. The cells were stained with 1:500 rat anti-HA (Roche #11867423001), followed by 1:1000 goat anti-rat 594 (Invitrogen #A11007), followed by a mixture of 1:500 mouse anti-ROP1 (Abnova #MAB17504) and 1:1000 rabbit anti-T. gondii (Abcam #ab138698), and finally with a mixture of 1:1000 goat anti-mouse 488 (Invitrogen #A11029), 1:1000 goat anti-rabbit 647 (Invitrogen #A21244), and 5 μg/mL DAPI (Sigma), each for 1h at room temperature. Images were acquired on a Nikon Ti-E inverted widefield fluorescence microscope with a Nikon CFI APO TIRF 100x/1.49 objective and Hamamatsu C11440 ORCA Flash 4.0 camera running NIS Elements (Nikon).
6
Quantifying Parasite Ubiquitination in Macrophages
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150,000 BMDMs per well were seeded in an 8-well μ-slide and stimulated with 10 ng/mL (~100 U/mL) recombinant mouse IFNγ for 24 h prior to infection. The BMDMs were infected with parasite strains at an MOI of 0.3 for 3 h, then washed and fixed with 4% w/v formaldehyde for 15 min. Prior to permeabilisation, the cells were blocked with 2% w/v BSA for 1 h and extracellular parasites were stained with 1:1000 rabbit anti-T. gondii (Abcam #ab138698) for 1 h at room temperature followed by 1:1000 goat anti-rabbit 405 (Invitrogen #A31556) for 1 h at room temperature. The cells were then permeabilsed with 0.2% v/v Triton X-100 for 15 minutes, blocked again with 2% w/v BSA for 1 h, stained with 1:200 mouse anti-ubiquitinylated proteins (Sigma #04–263) overnight at 4°C, and finally stained with 1:1000 goat anti-mouse 488 (Invitrogen #A11029) for 1 h at room temperature. Nine tiled fields of view were captured for each well on a Nikon Ti-E inverted widefield fluorescence microscope as above. The images were blinded, and the percentage of ubiquitinated vacuoles was determined manually using ImageJ, excluding T. gondii cells which were positive for extracellular staining. A median of 290 vacuoles were analysed per strain per replicate. Differences between strains were determined by two-sided t-test with Benjamini-Hochberg adjustment.
7
Measuring Ubiquitin Recruitment in Toxoplasma Infection
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Host cells were seeded and pre-stimulated as above in 96-well imaging plates (Ibidi). Cells were infected with 80,000 parasites/well and centrifuged at 300 g for 5 min to synchronise infection. At 3 h post-infection, cells were washed 3× in DPBS to remove extracellular parasites, then fixed in 4% PFA for 15 min and blocked with 3% BSA in DPBS for 1 h at room temperature. Extracellular parasites were stained prior to permeabilisation using 1:1,000 rabbit anti-toxo (Abcam, ab138698) and 1:500 goat anti-rabbit-AlexaFluor647 (Life Technologies, A21244). Cells were then permeabilised with 0.2% Triton-X100 for 10 min and re-blocked with 3% BSA. Host marker recruitment was probed for 2 h at room temperature using the following antibodies: mouse anti-total ubiquitin (1:200, Merck, ST1200), rabbit anti-K63 ubiquitin (1:100, Merck, 05–1308), rabbit anti-K48 ubiquitin (1:500, Sigma, ZRB2150), rabbit anti-M1 linear ubiquitin (1:200, Sigma, ZRB2114), or rabbit anti-RNF213 (1:1,000, Sigma, Human Protein Atlas no. HPA003347), followed by donkey anti-mouse-AlexaFluor488 (Thermo, A32766) or donkey anti-rabbit-AlexFluor488 (Thermo, A32790) for 1 h at room temperature. For measurement of K63 recruitment, cells were imaged on a Nikon Ti-E inverted widefield fluorescence microscope with a Nikon Plan APO 40×/0.95 objective, with at least 100 intracellular vacuoles scored/condition.
8
Quantifying Host c-Myc Activation in Toxoplasma Infection
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HFFs were grown to confluence in 8-well ibidi μ-slides (Ibidi, 80806). Cells were infected with 30,000 parasites in DMEM with 0.1% FBS. After 24 h, slides were fixed in 4% PFA for 15 min, permeabilised in 0.2% Triton-X100 for 10 min, then blocked in 3% BSA for 30 min. Host c-Myc was stained using 1:800 rabbit anti-cMyc for 2 h at room temperature, followed by 1:1,000 anti-Rabbit-AlexaFluor488 and 5 μg/ml DAPI for 1 h at room temperature. Images were acquired on a Nikon Ti-E inverted widefield fluorescence microscope with a Nikon Plan APO 40×/0.95 objective and Hamamatsu C11440 ORCA Flash 4.0 camera. Host nuclear c-Myc signal of infected cells was measured in FIJI, and the median background c-Myc signal was subtracted for each image. A minimum of 100 infected cells were analysed per strain for each biological replicate. Data is shown as the median fluorescence intensity for each strain relative to RHΔUPRT in each biological replicate. Differences between strains were determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test.
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