Axiovert fluorescence microscope

Manufactured by Zeiss

Sourced in Germany, United States

The Axiovert fluorescence microscope is a laboratory equipment designed for optical microscopy. It utilizes fluorescence techniques to visualize and analyze samples. The device features specialized optics and illumination systems to enable the observation of fluorescently labeled specimens.

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Lab products found in correlation

Lsm 710 confocal microscope, by Zeiss (6 mentions) Lsm 700, by Zeiss (4 mentions) Fm4 64, by Thermo Fisher Scientific (3 mentions) Lsm 780 confocal microscope, by Zeiss (3 mentions) Dm6000 upright confocal microscope, by Leica camera (2 mentions) Zen 2008, by Zeiss (2 mentions) Apotome, by Zeiss (2 mentions) 2030 reader, by PerkinElmer (1 mentions) Victor x3, by PerkinElmer (1 mentions) Rnase 3, by New England Biolabs (1 mentions) Rnase a t1 mix, by Thermo Fisher Scientific (1 mentions) Anti mouse alexa fluor 488, by Thermo Fisher Scientific (1 mentions) Anti stat1, by Cell Signaling Technology (1 mentions) Alexa 594 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions) Kaiser s glycerin gelatin, by Merck Group (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Calcein am, by Thermo Fisher Scientific (1 mentions) Propidium iodide, by Merck Group (1 mentions) Noble agar, by BD (1 mentions) Digital camera, by Nikon (1 mentions)

48 protocols using axiovert fluorescence microscope

Analyzing DDR2-Fc Binding and Modulation of Hydroxyapatite Formation

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Binding of DDR2-FC to HAP was analyzed by incubating 5mg of HAP powder with 200 μl of DDR2-Fc (100 μg/ml) in PBS overnight at 4°C on a turn-table[32 (link)]. Thereafter, the samples were centrifuged at 14000 rpm for 10 minutes to sediment the HAP and subjected to an immunostaining protocol. Briefly, the HAP slurry was washed three times in PBS and then incubated with anti-Fc antibody followed by washing and incubation with Alexa 488-conjugated secondary antibody. After final wash steps, an aliquot of the resulting particles was dispersed on a microscope slide, cover slipped and imaged using a Zeiss Axiovert fluorescence microscope.
To monitor if DDR2-Fc modulates the nucleation and growth of HAP independent of collagen, 200 μl of PBS, mSBF or PILP solutions (with or without 100 μg/ml of DDR2-Fc) were incubated in a 96-well microplate at 37°C in a CO₂ incubator with 95% relative humidity[32 (link)]. The absorbance of the wells (at 595 nm) was monitored using a microplate reader (Perkin Elmer 2030 reader, Victor X3) for up to 21 days. After 21 days, the supernatant was removed from the wells and the residual mineral deposits in the bottom of wells were retrieved by washing the wells with a 200 μl aliquot of de-ionized water. The resulting wash solutions were spotted on SEM stubs, air dried, gold-coated and imaged using SEM.

Farzadi A., Renner T., Calomeni E.P., Presley K.F., Karn N., Lannutti J., Dasi L.P, & Agarwal G. (2019). Modulation of biomimetic mineralization of collagen by soluble ectodomain of discoidin domain receptor 2. Materials science & engineering. C, Materials for biological applications, 104, 109905.

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Immunofluorescence Assay with RNase Treatment

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Cells were seeded onto glass coverslips, and transfected/infected cells were fixed with 4% formaldehyde for 15 min and permeabilized with 0.5% Triton X-100. For RNase treatments cells were incubated in RNase buffer (10 mM Tris:HCl [pH 8.3], 10 mM MgCl₂, 1 mM DTT, 60 mM NaCl) containing 50 U/ml RNase III (E6146S; New England Biolabs) or 50 μl/ml RNaseA/T1 mix (AM2286; Ambion), reflecting 25 U/ml and 1,000 U/ml, respectively, for 15 min at 37°C. Samples were then blocked in 4% FBS, incubated with primary antisera, and incubated with anti-mouse AlexaFluor 488 (A11029; Life Technologies) secondary antibody for 1 hr at room temperature. DNA was stained with 4′,6′-diamidino-2-phenylindole (DAPI). The fluorescent images were collected with a Zeiss LSM710 confocal microscope or a Zeiss Axiovert fluorescence microscope, using Zen 2008 software (Zeiss).

Burgess H.M, & Mohr I. (2015). Cellular 5′-3′ mRNA Exonuclease Xrn1 Controls Double-Stranded RNA Accumulation and Anti-Viral Responses. Cell Host & Microbe, 17(3), 332-344.

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DUSP1 Silencing Impact on STAT1 Translocation

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To assess the effect of DUSP1 silencing on translocalization of STAT1, cells expressing LV-cont or LV-shDUSP1 were seeded at a density of 2 × 10⁵ cells per plate. After 1 day of seeding, cells were fixed with 4% paraformaldehyde for 30 min, washed three times with PBS for 5 min, and incubated at 4°C overnight with a 1:50 dilution of anti-STAT1 (Cell Signaling Technology, Beverly, MA) in blocking solution (1% bovine serum albumin). Cells were washed three times with PBS, then incubated with Alexa 594-conjugated anti-rabbit IgG (Molecular Probes, Eugene, OR) for 1 h at room temperature. Nuclei were detected by staining with 1 μg/mL 4,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for 5 min. After three washes with PBS, preparations were mounted with Kaiser’s glycerin gelatin (Merck, Darmstadt, Germany) and examined using an Axiovert fluorescence microscope (Carl Zeiss, Göttingen, Germany).

Choi J.E., Kwon J.H., Kim J.H., Hur W., Sung P.S., Choi S.W, & Yoon S.K. (2015). Suppression of Dual Specificity Phosphatase I Expression Inhibits Hepatitis C Virus Replication. PLoS ONE, 10(3), e0119172.

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Melanoma Spheroid Invasion Assay

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2.5 × 10³ SKMel19 cells were cultured on 1.5 % noble agar (Difco/BD, Heidelberg, Germany) coated 96well plates to form spheroids within 3 days. For overexpression of CK1 isoforms either 2 μg/ml doxycycline were added on the second day or the medium was supplemented with the adenovirus. After 3 days spheroids were embedded into 1 mg/ml collagen I (Corning/BD, Heidelberg, Germany) diluted in complete growth medium and cultured for four more days. In case of treatment inhibitors were added to the medium. Daily microphotographs were taken and the area of the spheroids was measured using ImageJ and normalized to the size at day 0 after collagen embedding for the evaluation of tumor cell invasion into the collagen matrix. After 4 days spheroids were stained using 1 μM calcein-AM (Life technologies, Darmstadt, Germany) and 100 ng/ml propidium iodide (Sigma, Taufkirchen, Germany) for fluorescence live-dead staining of the melanoma cells. Fluorescence was detected with an Axiovert fluorescence microscope (Zeiss, Jena, Germany). Mean fluorescence intensities of the red channel were used to determine relative cell death induction.

Sinnberg T., Wang J., Sauer B, & Schittek B. (2016). Casein kinase 1α has a non-redundant and dominant role within the CK1 family in melanoma progression. BMC Cancer, 16, 594.

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Quantifying Cell Migration Using Scratch Assay

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HT29 cells stably expressing G0S2 shRNAs or non-silencing control (sh-NS) were plated to create a confluent monolayer on a 6 well plate. The monolayer was scratched with a p200 pipet tip and the edges were smoothed by washing the cells once with growth medium. Cells were cultured at 37°C and time lapse photographs taken over two days at several points in each scratch using a Zeiss Axiovert fluorescence microscope. The results are represented as percentage of distance invaded. If width of the scratch at day 0 = D0, width of scratch at day 2 = D2, then percentage of distance invaded = D0-D2/D0 × 100. For the ATGListatin experiment, the scratch was made in a confluent HT29 cells grown on a 6 well plate. 100 μM of ATGListatin or DMSO (control) was added to the cells and cultured for four days.

Zagani R., El-Assaad W., Gamache I, & Teodoro J.G. (2015). Inhibition of adipose triglyceride lipase (ATGL) by the putative tumor suppressor G0S2 or a small molecule inhibitor attenuates the growth of cancer cells. Oncotarget, 6(29), 28282-28295.

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Visualizing NET Formation in Neutrophils

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Mouse bone marrow neutrophils were isolated from WT or PLD2^−/− mice. Neutrophils (10⁵ cells) were seeded on 12-mm 0.01% poly-l-lysine–coated coverslips in 24-well plates and were stimulated with ionomycin (5 µM). Cells were fixed with 4% paraformaldehyde and then stained with SYTOX Green nucleic acid stain (5 µM). NETs were visualized on an Axiovert fluorescence microscope (Carl Zeiss) and images were taken using a Nikon digital camera.

Lee S.K., Kim S.D., Kook M., Lee H.Y., Ghim J., Choi Y., Zabel B.A., Ryu S.H, & Bae Y.S. (2015). Phospholipase D2 drives mortality in sepsis by inhibiting neutrophil extracellular trap formation and down-regulating CXCR2. The Journal of Experimental Medicine, 212(9), 1381-1390.

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Morphological Changes after BIIB021 Treatment

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To detect the morphological changes following treatment with BIIB021 (0, 100, 200 and 400 nM), SKM-1 cells were plated at an initial density of 1×10⁵ cells/well in a 24-well plate and treated with BIIB021 for 24 h. The treated cells were fixed with 3.7% paraformaldehyde for 30 min and then stained with Hoechst 33258 (0.5 μg/ml) for 20 min at room temperature. The cells were counted under an Axiovert fluorescence microscope (Carl Zeiss, Göttingen, Germany) with an excitation wavelength of 350 nm and an emission wavelength of 460 nm.

LIN S., LI J., ZHOU W., QIAN W., WANG B, & CHEN Z. (2014). BIIB021, an Hsp90 inhibitor, effectively kills a myelodysplastic syndrome cell line via the activation of caspases and inhibition of PI3K/Akt and NF-κB pathway proteins. Experimental and Therapeutic Medicine, 7(6), 1539-1544.

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Osteogenic Differentiation of Cells

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The cells were seeded at a density of 4 × 10⁴ cells in 500 μL simple osteogenic medium/well in Nunc Lab-Tek 4 wells chamber slides. After 10 days of cultivation, the cells were washed 3 times with Phosphate Buffered Saline (PBS) and fixed with 4% paraformaldehyde solution for 20 min at room temperature. The cell permeabilization was performed with 0.1% Triton X-100 for 20 min, followed by blocking of unspecific antibody bound with 10% Bovine Serum Albumin (BSA) for 20 min at room temperature. Staining with primary antibodies was performed for bone cells markers: collagen 1A, osteopontin, and osteocalcin (all from Santa Cruz Biotechnologies) at a dilution of 1:50. Samples were incubated overnight at 4 °C in dark, followed by incubation 45 min at room temperature with secondary antibody goat anti-mouse IgG labeled with FITC (Santa Cruz Biotechnologies) (1:50). Slides were mounted with 4,6-diamidino-2-phenylindole (DAPI) (Santa Cruz Biotechnologies) for nuclei staining. Each step was followed by 3 washes with PBS. The samples were visualized with a Zeiss Axiovert fluorescence microscope using filters at 488, 546 and 340/360 and images were taken with an AxioCam MRC camera.

Petrescu N.B., Jurj A., Sorițău O., Lucaciu O.P., Dirzu N., Raduly L., Berindan-Neagoe I., Cenariu M., Boșca B.A., Campian R.S, & Ilea A. (2020). Cannabidiol and Vitamin D3 Impact on Osteogenic Differentiation of Human Dental Mesenchymal Stem Cells. Medicina, 56(11), 607.

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Immunofluorescence and RNA-FISH in Mouse Embryos

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After zona pellucida removal, embryos were fixed in 3% PFA for 10 min on coverslips then permeabilized (Triton/VRC/PBS) on ice for 15 min. Embryos were then blocked in PBS/BSA for 15 min at RT, incubated with primary antibodies with RNAase inhibitor (1–2 h at RT) and then secondary antibodies (30 min at RT)^{5 (link)}. Embryos were then again fixed in 4% PFA for 10 min at RT. RNA-FISH was then performed as mentioned previously. Images were acquired using inverted laser scanning confocal microscope with spectral detection (LSM700—Zeiss) equipped with a 260 nm laser (RappOpto), with a ×60 objective and 0.2 μm Z-sections or a confocal wide-field Deltavision core microscope (Applied Precision—GE Healthcase) with a ×60 objective (1.42 oil PL APO N) and 0.2 μm Z-sections or a 200 M Axiovert fluorescence microscope (Zeiss) equipped with an ApoTome was used to generate 3D optical sections. Sequential z-axis images were collected in 0.3 μm steps. Images were analysed using ImageJ software (Fiji, NIH).
ICMs obtained from Utx^FDC/wt females were PCR-genotyped after image acquisition (details available upon request).
All the antibodies and probes used in this study are listed in Supplementary Table 1 along with the information on dilution ratios.

Borensztein M., Okamoto I., Syx L., Guilbaud G., Picard C., Ancelin K., Galupa R., Diabangouaya P., Servant N., Barillot E., Surani A., Saitou M., Chen C.J., Anastassiadis K, & Heard E. (2017). Contribution of epigenetic landscapes and transcription factors to X-chromosome reactivation in the inner cell mass. Nature Communications, 8, 1297.

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Immunofluorescence Staining Protocol

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The culture medium was aspirated, cells washed once in PBS, then fixed in 4% PFA in PBS for 15 minutes at room temperature, followed by three PBS washes of 3 min each. Cells were permeabilised and blocked for 1 hour (2% BSA, 0.3% Triton x100 in PBS). Cells were washed in PBS and incubated with primary antibodies overnight at 4 °C. Cells were washed in PBS (3x, 15 min) and incubated with secondary antibodies for 2 hrs at room temperature. Cells were washed in PBS (3x, 15 minutes) before imaging in PBS in the wells. Images were taken using a Zeiss Axiovert fluorescence microscope. Working dilutions of the antibodies were as follows: anti-HMOX1, 1:100; anti-mCherry, 1:100; anti-Oct3/4, 1:100; anti-Pax2, 1:200; anti-LHX1, 1:100; anti-CALB, 1:200; anti-WT1, 1:200; anti-CDH1, 1:300; anti-NPHS1, 1:200; anti-BRACHYURY, 1:100. Details of the used antibodies are listed in the key resources table. Antibody solutions were prepared using PBS containing 1% BSA.

Lawrence M.L., Elhendawi M., Morlock M., Liu W., Liu S., Palakkan A., Seidl L.F., Hohenstein P., Sjögren A.K, & Davies J.A. (2022). Human iPSC-derived renal organoids engineered to report oxidative stress can predict drug-induced toxicity. iScience, 25(3), 103884.

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