Axio observer z1 inverted fluorescence microscope

Manufactured by Zeiss

Sourced in Germany

The Axio Observer Z1 is an inverted fluorescence microscope designed for advanced imaging applications. It features a stable and ergonomic stand with a large working distance and a high-quality optical system. The microscope is capable of various contrast methods, including brightfield, phase contrast, and fluorescence imaging.

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Lab products found in correlation

Zen 2011, by Zeiss (1 mentions) Axiocam mrm, by Zeiss (1 mentions) Volocity software, by PerkinElmer (1 mentions) Roti immunoblock, by Carl Roth (1 mentions) Alexa fluor 488, by Thermo Fisher Scientific (1 mentions) Dabco, by Carl Roth (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Carl Roth (1 mentions) Alexa fluor 488 and, by Thermo Fisher Scientific (1 mentions) Normal donkey serum (nds), by Thermo Fisher Scientific (1 mentions) Alexa fluor 546 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions) Hcs dna damage kit, by Thermo Fisher Scientific (1 mentions) Pkh 26 cell linker kit, by Merck Group (1 mentions) Dapi 4 6 diamidino 2 phenylindole, by Thermo Fisher Scientific (1 mentions) Ab4055, by Abcam (1 mentions) Caspase 3, by Cell Signaling Technology (1 mentions) Rorgt, by Thermo Fisher Scientific (1 mentions) Foxp3, by Abcam (1 mentions) Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions) Mowiol, by Merck Group (1 mentions) Matrigel, by Corning (1 mentions)

22 protocols using axio observer z1 inverted fluorescence microscope

EGFP Expression in Komagataella phaffii

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Komagataella phaffii cells expressing EGFP were grown in 5 mL BMGY for 16 h at 28 °C. After cell count, pelleted cells were re-suspended in 20 mL BMMY to a final OD₆₀₀ of 0.3. The culture was incubated at 28 °C and methanol was added to a final concentration of 0.5%. After 24 h of induction cells were imaged in a Zeiss Axio Observer Z1 Inverted Fluorescence Microscope equipped with 63× NA 1.4 oil immersion objective and a cooled CCD camera to analyze EGFP fluorescence. The images were acquired with Zen2011 software (Zeiss) and manipulated with Microsoft Office Picture Manager or Adobe Photoshop.

Betancur M.O., Reis V.C., Nicola A.M., De Marco J.L., de Moraes L.M, & Torres F.A. (2017). Multicopy plasmid integration in Komagataella phaffii mediated by a defective auxotrophic marker. Microbial Cell Factories, 16, 99.

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Quantifying Microtubule Dynamics in Cells

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Cells successfully expressing GFP were chosen and analyzed with an Axio observer Z1 inverted fluorescence microscope equipped with an Axiocam Mrm, Incuabator X1multi S1, TempModul S1, and CO₂ Modul S1 (all from Zeiss). Images were taken every 1s at an exposure time of 600–800 ms with a 488 nm laser for 15–20 min with 100× objective. All the measurements were performed in a humidified atmosphere at 37 °C and at 5% CO₂. Quantitative analysis of the microtubule dynamics was carried out on time-lapse movies of cells expressing EB1-GFP. Microtubule growth rates were obtained by tracking EB1-GFP comets at microtubule plus-ends. Images were recorded and movies were assembled by means of AxioVision software. More than 750 MT tips in Fibroblast cells and nearly 800 MT tips in axons of human neurons were analyzed.

Shaebani M.R., Pasula A., Ott A, & Santen L. (2016). Tracking of plus-ends reveals microtubule functional diversity in different cell types. Scientific Reports, 6, 30285.

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Spatial Analysis of Scaffold Fluorescence

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Immunostained samples were imaged at 0.5 mm intervals in both the horizontal and vertical directions from the center of the scaffold to the periphery using an Axio Observer.Z1 Inverted Fluorescence Microscope (Carl Zeiss). Samples were imaged in both the 488 and 546 channels with the same exposure settings used for each position on the same scaffold. Intensity of the fluorescence was quantified using ImageJ and the ratio of the intensity at 488/546 at each position from the center was averaged and plotted.

Kador K.E., Alsehli H.S., Zindell A.N., Lau L.W., Andreopoulos F.M., Watson B.D, & Goldberg J.L. (2014). Retinal Ganglion Cell Polarization Using Immobilized Guidance Cues on a Tissue-Engineered Scaffold. Acta biomaterialia, 10(12), 4939-4946.

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In Vivo FITC-c-CPE Tumor Cell Imaging

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C.B-17/SCID mice developing ascites were generated following IP injection of 5 ×10⁶ OSPC-ARK-1-derived cells. When ascitic fluid was abundant in the abdomen (i.e., 10–12 weeks later), animals were injected with 10μg of FITC-c-CPE IP and 12 hours later, immunofluorescence was performed on Ficoll-Hypaque separated cells isolated from ascites. Briefly, mice were anesthetized and abdominal paracentesis was performed. Cell pellet was isolated through a Ficoll gradient and an anti-human CD326 (EpCAM) PE conjugated antibody (#12-9326-42 eBioscience, San Diego, CA) was used to label the tumor cells. Images were captured using an Axio Observer.Z1 inverted fluorescence microscope (Zeiss, Germany) installing a X-cite 120Q illuminator (Lumen Dynamics, UK) and analyzed with Volocity software (Improvision, Perkin Elmer, MA). Cells isolated from the ascites of mice not injected with FITC-c-CPE were used as a control.

Cocco E., Shapiro E.M., Gasparrini S., Lopez S., Schwab C.L., Bellone S., Bortolomai I., Sumi N.J., Bonazzoli E., Nicoletti R., Deng Y., Saltzman W.M., Zeiss C.J., Centritto F., Black J.D., Silasi D.A., Ratner E., Azodi M., Rutherford T.J., Schwartz P.E., Pecorelli S, & Santin A.D. (2015). Clostridium Perfringens Enterotoxin C-terminal domain labeled to fluorescent Dyes for in vivo visualization of micro-metastatic chemotherapy-resistant ovarian cancer. International journal of cancer. Journal international du cancer, 137(11), 2618-2629.

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Immunocytochemical Visualization of PMEL

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For immunocytochemistry, MAC-T cells were seeded on coverslips. After overnight culture growth, coverslips were washed three times with PBS and fixed with ice-cold methanol (10 min, −20 °C). Anti-PMEL (1:100) primary antibody was incubated overnight at 4 °C. Unspecific antibody binding was blocked with Roti-Immunoblock (Carl Roth GmbH). Goat anti-rabbit IgG secondary antibody conjugated with Alexa Fluor® 488 (1:1000; # A11034, Life Technologies, Germany) was used to detect specific binding of the primary antibody. Nuclei were counterstained with 600 nM DAPI (Carl Roth GmbH). The same protocol, excluding incubation with the primary antibody, was used for the negative controls. Cells were mounted with DABCO (Carl Roth GmbH) and analyzed with the Axio Observer.Z1 inverted fluorescence microscope (Carl Zeiss Microscopy GmbH) using the Plan-Apochromat 63x/1.4 Oil DICIII objective.

Knaust J., Weikard R., Albrecht E., Brunner R.M., Günther J, & Kühn C. (2020). Indication of Premelanosome Protein (PMEL) Expression Outside of Pigmented Bovine Skin Suggests Functions Beyond Eumelanogenesis. Genes, 11(7), 788.

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Quantifying Doxorubicin Uptake in CHRF Cells

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To compare DOX uptake by CHRF cells when administered freely or in NP formulations, 4 × 10⁴ CHRF cells was seeded per well of an 8-well chamber plate in PMA-supplemented media for 24 h. Cells were then treated with free DOX, DOX-loaded PEG-PLGA NPs, or DOX-loaded MWNPs at 1.5 μM. After 4 h of treatment, cells were washed twice with PBS to remove media and excess/non-internalized DOX. Cells were fixed with 4% formaldehyde for 10 min and then washed twice with PBS. Following fixation, the chambers were removed, and a glass cover slip was sealed onto the glass slide for imaging. Images were acquired at 40X magnification on a Zeiss Axioobserver Z1 inverted fluorescence microscope. An increased fluorescent DOX signal was taken to indicate more DOX uptake.
Cellular fluorescence in the images was analyzed using ImageJ. Five background values were captured for each image, and cells were outlined with the freehand selection tool. The area, integrated density, and mean gray value were measured for each selection, and the corrected total cell fluorescence (CTCF) was calculated using the following formula:

CTCF = integrated density - (area of selected cell \times mean fluorecence of background)

Harris J.C., Sterin E.H, & Day E.S. (2022). Membrane-Wrapped Nanoparticles for Enhanced Chemotherapy of Acute Myeloid Leukemia. ACS biomaterials science & engineering, 8(10), 4439-4448.

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Immunofluorescence Staining and DNA Methylation Protocol

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Samples collected for immunofluorescence staining at the indicated time points were washed once with PBS and fixed in 4% paraformaldehyde for 15 min. Samples were washed three times with PBS for 5 min each and permeabilized using 0.5% Triton X‐100 for 10 min. After three subsequent PBS washes, samples were blocked with 5% normal donkey serum (NDS; Jackson Immunoresearch, 01 700 0121) in PBS for 1 h. Samples were incubated with primary antibodies (Refer to Table S1, Supporting Information) in antibody dilution buffer (1% NDS + 0.1% Triton X‐100 in PBS) for either 1 h or overnight at 4 °C followed by three PBS washes and a 1 h incubation with Alexa Fluor 488‐ and/or Alexa Fluor 546‐conjugated secondary antibodies (Molecular Probes). Nuclei were stained with DAPI in PBS for 10 min. For co‐staining of EdU with other markers, samples were first stained for EdU followed by the immunostaining procedure from the blocking step. Epifluorescence images were collected using a Zeiss Axio Observer Z1 inverted fluorescence microscope and analyzed using ImageJ.
For DNA methylation staining, samples were fixed with ice‐cold 70% ethanol for 5 min followed by three PBS washes. Samples were then treated with 1.5m HCl for 30 min and washed thrice with PBS. The immunostaining procedure proceeded from the donkey serum blocking step as aforementioned.

Song Y., Soto J., Wang P., An Q., Zhang X., Hong S., Lee L.P., Fan G., Yang L, & Li S. (2021). Asymmetric Cell Division of Fibroblasts is An Early Deterministic Step to Generate Elite Cells during Cell Reprogramming. Advanced Science, 8(7), 2003516.

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Immunofluorescence Staining of Cultured Cells

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Cells in 24-well plates were rinsed twice with PBS, fixed in 4% paraformaldehyde for 20 minutes at room temperature, and rinsed twice more with PBS. Then, cells were permeabilized with 0.3% Triton X-100 in PBS for 5 minutes, followed by blocking with 3% BSA for 1 hour at room temperature with gentle shaking. After this, cells were incubated with the indicated primary antibody overnight at 4°C, rinsed in the morning with PBS for 2 cycles (10 minutes each), and incubated with fluorophore-conjugated secondary antibody for 1 hour at room temperature in the dark. Cells were then finally stained with 10 μg/ml Hoescht 333342 for 10 minutes, rinsed 3 times with PBS, and imaged on an AXIO Observer.Z1 inverted fluorescence microscope (Zeiss, Oberkochen, Baden-Württemberg, Germany).

Mitchell W., Goeminne L.J., Tyshkovskiy A., Zhang S., Chen J.Y., Paulo J.A., Pierce K.A., Choy A.H., Clish C.B., Gygi S.P, & Gladyshev V.N. (2023). Multi-omics characterization of partial chemical reprogramming reveals evidence of cell rejuvenation. bioRxiv.

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Notch-1 Expression in TNBC and Breast Epithelial Cells

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Notch-1 receptor expression in MDA-MB-231 TNBC cells and MCF-10A breast epithelial cells was analyzed using immunocytochemistry staining. Cells were plated at 2.5 × 10⁴ cells per well (MDA-MB-231 cells) or 1.0 × 10⁴ cells per well (MCF-10A cells) in a 24-well plate and incubated for approximately 48 h. The cells were then fixed in 4% formaldehyde prior to quenching peroxidase reactions with 3% peroxide. The samples were then rinsed with PBS and blocked with 3% bovine serum albumin in PBS for 1 h. After blocking, the cells were then rinsed and incubated in primary anti-human Notch-1 antibody (Santa Cruz; 1 μg/mL) for 1 h at room temperature. The samples were subsequently rinsed three times in PBS and incubated in secondary HRP-conjugated goat anti-rabbit IgG antibody (Pierce; 0.8 μg/mL) for 40 min at room temperature. The cells were rinsed three times in PBS and developed in 3-amino-9-ethylcarbazole (AEC) for 15 min. Finally, the cells were rinsed in PBS and imaged on a Zeiss Axioobserver Z1 Inverted Fluorescence Microscope.

Valcourt D.M., Dang M.N., Scully M.A, & Day E.S. (2020). Nanoparticle-Mediated Co-Delivery of Notch-1 Antibodies and ABT-737 as a Potent Treatment Strategy for Triple-Negative Breast Cancer. ACS nano, 14(3), 3378-3388.

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DNA Damage Assay in Fibroblasts

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After cells passed through the microdevice, 5 × 10³ fibroblasts were plated and allowed to attach for 3 hours in a 96-well plate. DNA damage assays were performed using the HCS DNA Damage Kit (Invitrogen, H10292) according to the manufacturer’s protocol. Cells were fixed with 4% paraformaldehyde solution for 15 minutes at room temperature and permeabilized by 0.25% Triton X-100 in PBS for another 15 minutes at room temperature. Cells were washed three times with PBS and incubated in 1% bovine serum albumin (BSA) solution for 1 hour, followed by phospho-H2AX (pH2AX) antibody (1:1,000) for 1 hour at room temperature and then Alexa Fluor 555 goat anti-mouse IgG (H+L) secondary (1:5,000) with Hoechst 33342 (1:6,000) for another 1 hour at room temperature after removing the antibody. Epifluorescence images were collected using a Zeiss Axio Observer Z1 inverted fluorescence microscope and analysed using ImageJ. Results were normalized to control samples (that is, cell passing through channels of >200 μm), and cells treated with 200 nM lipopolysaccharide served as a positive control.

Song Y., Soto J., Chen B., Hoffman T., Zhao W., Zhu N., Peng Q., Liu L., Ly C., Wong P.K., Wang Y., Rowat A.C., Kurdistani S.K, & Li S. (2022). Transient nuclear deformation primes epigenetic state and promotes cell reprogramming. Nature materials, 21(10), 1191-1199.

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