Cells were grown on 8-well chamber slides (Labtek, Thermo Fisher Scientific), precoated for 1 h with fibronectin (10 μg/ml) for U87-MG or with poly-DL-ornithine (Sigma-Aldrich) (10 µg/ml) for GBM6, to be treated for 6 h with ProA, digoxin, bufalin or digitoxin. As previously described16 (link), cells were incubated with the anti-EB1 (clone 5; BD Biosciences, San Jose, CA) and α-tubulin (clone DM1A; Sigma-Aldrich) primary antibodies, and then with Alexa488 or 568-conjugated secondary antibodies (Invitrogen). Staining was observed using either a Leica DM-IRBE microscope or a Leica TCS SP5 confocal laser-scanning microscope (Leica, Heidelberg, Germany). Images were acquired using Metamoph software or the Leica Confocal software, and were processed using Image J software. For each experimental condition, at least 100 EB1 comets (in at least 10 cells) were examined to measure their length.
Leica dm irbe microscope
Manufactured by Leica camera
Sourced in Germany, South Africa
The Leica DM-IRBE is a research-grade inverted microscope designed for advanced microscopy applications. It features a modular design and can be configured with a range of objectives, illumination systems, and camera options to suit various experimental requirements. The DM-IRBE provides high-quality imaging and analysis capabilities for a wide variety of samples and applications.
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Lab products found in correlation
Lab tek, by Thermo Fisher Scientific (3 mentions)
Anti eb1 clone 5, by BD (2 mentions)
Poly dl ornithine, by Merck Group (2 mentions)
α tubulin clone dm1a, by Merck Group (2 mentions)
Leica tcs sp5 confocal laser scanning microscope, by Leica camera (2 mentions)
Vectashield mounting medium with 4 6 diamidino 2 phenylindole dapi, by Vector Laboratories (2 mentions)
Alexa488 or 568 conjugated secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Alexa 488 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti α tubulin clone dm1a, by Merck Group (1 mentions)
Dako mounting media, by Agilent Technologies (1 mentions)
Zoletil 100, by Virbac (1 mentions)
Leica tcs sp5 confocal system, by Leica Microsystems (1 mentions)
Leica dm irbe microscope, by Leica Microsystems (1 mentions)
Leibovitz s l 15 medium, by Thermo Fisher Scientific (1 mentions)
Sk n sh, by Merck Group (1 mentions)
Leica confocal software, by Leica camera (1 mentions)
8 well chamber slide, by Thermo Fisher Scientific (1 mentions)
Evos m5000 imaging system, by Thermo Fisher Scientific (1 mentions)
Vectashield antifade mounting medium with dapi, by Vector Laboratories (1 mentions)
Cd11b, by Thermo Fisher Scientific (1 mentions)
10 protocols using leica dm irbe microscope
1
Measuring EB1 Comet Length in GBM Cells
2
Immunofluorescence Staining of GBM6 Cells
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GBM6 cells were grown on 8-well chamber slides (Labtek, Thermo Fisher Scientific), precoated for 1 h with poly-DL-ornithine (Sigma-Aldrich) (10µg/ml), to be treated for 48 h with BAL27862. As previously described [19 (link)], cells were incubated with an anti- α-tubulin (clone DM1A; Sigma-Aldrich) primary antibody, and then with Alexa488 secondary antibody (Invitrogen). Staining was observed using either a Leica DM-IRBE microscope. Images were acquired using Metamoph software and were processed using Image J software.
3
Visualizing Protein Aggregation in Yeast
4
Hsp70 Distribution in Rat Brain After IV Injection
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To assess the distribution of Hsp70 labeled with Alexa Fluor® 555 in the brain following IV injection, rats were anesthetized by intraperitoneal injection with 10 mg Zoletil-100 (tiletamine hydrochloride and zolazepam; Virbac, Carros, France) and 0.2 mL 2% Rometar (xylazine hydrochloride; Bioveta, Ivanovice na Hané, Czech Republic). Following intracardial perfusion with 4% paraformaldehyde in 0.01 M phosphate-buffer solution, the brain was extracted to be examined for the presence of fluorochrome-labeled Hsp70. Each brain was embedded in TissueTek® and stored at −80°C. Serial cryosections were obtained in the coronal plane with a slice thickness of 10 μm. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Sections were mounted with DAKO mounting media (Agilent Technologies, Santa Clara, CA, USA). For analysis of the presence of annexin V in the brain tissues, we used a commercially available annexin V–fluorescein isothiocyanate kit (Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions. Brain cryosections were stained for the presence of annexin V and nuclei were detected using DAPI. Images were captured with a Leica TCS SP5 confocal system (Leica Microsystems, Wetzlar, Germany) at 488 or 543 nm or a Leica DM IRBE microscope at 405 nm.
5
Live-cell imaging of cyclin B1 dynamics
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For microinjection and microscopy, the cells were grown on a Bioptechs ΔT heating stage (Bioptechs, Butler, PA) attached to a Leica DMIRBE microscope and the culture medium was replaced with Leibovitz's L-15 medium (Life Technologies Ltd) supplemented with 10% foetal bovine serum, penicillin (100 U/ml) and streptomycin (100 µg/ml). Cells were microinjected with cDNA encoding cyclin B1-Venus at a concentration of 3 ng/µl in G2 cells, using a semiautomatic microinjector (Eppendorf, Stevenage, UK) on a Leica DMIRBE microscope (Leica Microsystems, Milton Keynes, UK) and assayed by time-lapse DIC and fluorescence microscopy as previously described (Karlsson and Pines, 1998 ). Parameters used for all images captured were exposure time 200 mseconds, 40× oil objective lens with a numerical aperture of 1.2. All images were captured at 3 minutes intervals and analyzed by SlideBook software (Intelligent Imaging Innovations, Denver, CO, USA).
6
Analyzing EB1 Comet Dynamics in Glioblastoma and Neuroblastoma Cells
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Cells were grown on 8-well chamber slides (Labtek, Thermo Scientific, Roskilde, Denmark), precoated for 1 hour with fibronectin (10 μg/ml) for U87-MG or with type I collagen (30µg/ml) for SK-N-SH (Sigma Aldrich), to be treated for 6 hours with MTAs and inhibitors. As previously described [32 (link)], cells were incubated with the anti-EB1 (clone 5; BD Biosciences, San Jose, CA) and α-tubulin (clone DM1A; Sigma Aldrich) primary antibodies, and then with Alexa488 or 568-conjugated secondary antibodies (Molecular Probes). Staining was observed using either a Leica DM-IRBE microscope or a Leica TCS SP5 confocal laser-scanning microscope (Leica, Heidelberg, Germany). Images were acquired using Metamoph software or the Leica Confocal software, and were processed using Image J software. For each experimental condition, at least 400 EB1 comets (in 40 cells) were examined to measure their length.
7
Visualizing Protein Aggregation in Yeast
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Microscopy samples were grown and induced as they were for immunoblotting. For TDP-43 samples (W303aΔhsp104-pAG303GAL-TDP-43-GFPS11-pAG305GAL-GFPS-10), cells were harvested, fixed in 1mL 70% ethanol, and immediately pelleted. The cells were then washed 3 times with cold PBS and resuspended in 15μL of Vectashield mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories). α-syn (W303aΔhsp104-pAG303GAL-α-syn-YFP-pAG304GAL-α-syn-YFP) and FUS (W303aΔhsp104-pAG303GAL-FUS-GFP) samples were imaged live. All cells were imaged at 100x magnification using a Leica-DMIRBE microscope. Analysis of cells was performed in ImageJ (Schneider et al., 2012 (link)). For each sample, 97-530 cells were quantified in two or three independent trials.
8
Microscopic Analysis of Yeast and HeLa Cells
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For yeast microscopy, W303aΔhsp104-pAG303GAL-FUSWT-GFP yeast were transformed with the indicated Kapβ2 variant or vector control. Microscopy samples were grown and induced as they were for immunoblotting. Prior to imaging, cells were treated with Hoechst 33342 stain (0.1 mg/ml). All cells were imaged at 100× magnification using a Leica-DMIRBE microscope or the EVOS M5000 Imaging System. Analysis of cells was performed in ImageJ (133 (link)). For each sample, at least 100 cells were quantified in at least three independent trials.
For HeLa cell microscopy, transfected HeLa cells were fixed with 2% formaldehyde for 30 min at room temperature, followed by treatment with Triton X-100 for 6 min to permeabilize cells. Coverslips were then assembled using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories) and sealed before imaging. Images were taken at 100× magnification using the EVOS M5000 Imaging System (ThermoFisher) and processed using ImageJ (133 (link)). At least 100 cells were counted for each condition across three independent trials.
For HeLa cell microscopy, transfected HeLa cells were fixed with 2% formaldehyde for 30 min at room temperature, followed by treatment with Triton X-100 for 6 min to permeabilize cells. Coverslips were then assembled using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories) and sealed before imaging. Images were taken at 100× magnification using the EVOS M5000 Imaging System (ThermoFisher) and processed using ImageJ (133 (link)). At least 100 cells were counted for each condition across three independent trials.
9
Immunofluorescence Staining of Cells
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Cells were seeded onto sterile glass coverslip in a 24 well plate and stimulated. Non-adherent cells were gently removed by pipetting, coverslips washed with PBS and fixed for 20 min with 4% formaldehyde (Sigma Aldrich). Cells were permeabilized for 5 min with 0.1% Triton-X100 (Fluka) and incubated for 30 min with PBS + 10% FBS (Sigma Aldrich) to reduce unspecific binding. Coverslips were incubated with primary antibodies (diluted in PBS + 5% FBS) overnight at 4°C in a humidified chamber. AlexaFluor−488, −546, or −647 conjugated anti-mouse, anti-rabbit or anti-rat secondary antibodies (Invitrogen) were incubated for 1 h at room temperature. Coverslips were washed and mounted on SuperFrost glass slides (Menzel) with Mowiol 40–88 mounting media containing 2.5% 1,4-diazobicyclo-[2,2,2]-octane (Sigma Aldrich). Images were acquired with a with a Leica DM IRBE microscope equipped with a TCS-SP confocal scanner head (with 488 nm Ar and 543–633 nm HeNe lasers) at a magnification of 63 × and processed with Fiji (ImageJ) software (18 (link)). Antibodies (clones and vendors where applicable): α-tubulin (DM1A, Sigma Aldrich), Candida (Abnova), CD11b (M1/70, Thermo Fisher), F4/80 (BM8, BioLegend), FcεR β chain (JRK b), LAMP-1 (1D4B, eBiosciences), TBP (SI-1, Santa Cruz).
10
Chemotaxis Assay of Fibroblasts
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We utilized Ibidi's 2-D µ-Slide chemotaxis assay system (80306; Ibidi, Planegg, Germany) according to the manufacturer's specifications using FBs cultured in DMEM. Human recombinant bFGF (100-18b; Peprotech, Rocky Hill, CT) was used at a final concentration of either 10 or 20 ng/mL. The recommended cell seeding density of 3 × 10 6 was observed. Migration was monitored over 24 h using standard inverted stage microscopy with an incubated stage at 100× magnification with 1 image recorded every 20 min via a Motic Moticam T microscope tablet (Motic, Hong Kong, China) camera connected to a Leica DMIRBE microscope (Leica, Wynberg, South Africa). A minimum of 26 cells were analyzed per experiment by manually tracking the cell paths using the chemotaxis and migration toolkit plug-in for ImageJ image processing software (www.ibidi. de/applications/ap_chemo.html).
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