Apoptosis was determined using cleaved caspase 3 IF as previously described [12 (link)]. Briefly 40,000 cells were seeded in 12 well plates on glass coverslips in triplicate. 24 hours later cells were treated with chemotherapeutic agents with or without A69 for 24 hours. After treatment, cells were fixed in 3.7% formaldehyde for 15 minutes and permeabilized in 0.3% Triton X-100 for 15 minutes. Antibodies were diluted in blocking buffer that consisted of 0.2% non-fat dry milk, 2% Bovine Serum Albumin and 0.3% Triton X-100 in phosphate buffered saline (PBS). Primary anti-cleaved caspase 3 rabbit monoclonal antibody (1:400, Cell Signaling Technology, Danvers, MA) was applied to cells for 1 hour at 37°C. Following washes in PBS, samples were incubated in goat-anti-rabbit Alexa Fluor 488 (1:1000, Life Technologies, Carlsbad, CA) and Alexa 555 conjugated Phalloidin (1:200, Life Technologies) to visualize F-actin. Slides were mounted with Fluoromount G with Hoescht (Sigma) to label cell nuclei. The percentage of positive cells was determined for each assay with at least 150 cells being counted per condition using the counting feature on an Evos Xl core microscope. Each assay was run in triplicate and repeated three times. Representative images were taken on Zeiss Axio A1 Microscope with an AxioCam MRc digital camera.
Axio a1 microscope
Manufactured by Zeiss
Sourced in Germany
The Axio A1 microscope is a compact and versatile optical microscope designed for routine observation and analysis. It features a sturdy and ergonomic construction, providing a stable platform for precise imaging. The microscope is equipped with a range of objective lenses, allowing for various magnification levels to suit different sample types and applications.
Automatically generated - may contain errors
Lab products found in correlation
Axiocam mrc5 ccd camera, by Zeiss (4 mentions)
Axiocam mrc, by Zeiss (4 mentions)
Alexa 555 conjugated phalloidin, by Thermo Fisher Scientific (2 mentions)
Fluoromount g, by Merck Group (2 mentions)
Masson s trichrome stain kit, by Merck Group (2 mentions)
Axiovision software, by Zeiss (2 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Vectashield medium, by Vector Laboratories (2 mentions)
Axiocam mrc digital camera, by Zeiss (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Titan g2 80 200, by Thermo Fisher Scientific (1 mentions)
Matrigel, by BD (1 mentions)
Polyclonal rabbit anti rat immunoglobulin hrp, by Agilent Technologies (1 mentions)
Rat anti mouse cd8, by BD (1 mentions)
Rat anti mouse foxp3, by Thermo Fisher Scientific (1 mentions)
Diaminobenzidine dab, by Agilent Technologies (1 mentions)
Mouse anti flag m2, by Merck Group (1 mentions)
Alexa fluor 594 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Rabbit anti v5, by Cell Signaling Technology (1 mentions)
19 protocols using axio a1 microscope
1
Quantifying Apoptosis using Cleaved Caspase-3 IF
2
Phospho-H2AX Assay in Cells
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40,000 cells were plated on glass coverslips in a 12 well plate in triplicate and 24 hours later cells were treated as described above. After treatment, cells were fixed in 3.7% formaldehyde for 15 minutes and permeabilized in 0.3% Triton X-100 for 15 minutes. Antibodies were diluted in blocking buffer that consisted of 0.2% non-fat dry milk, 2% Bovine Serum Albumin and 0.3% Triton X-100 in phosphate buffered saline (PBS). Primary anti-phospho H2AX (1:650, Millipore) was applied to cells for 1 hour at 37 °C. Following washes in PBS, samples were incubated in goat-anti-rabbit Alexa Fluor 488 (1:1000, Life Technologies) and Alexa 555 conjugated Phalloidin (1:200, Life Technologies) to visualize F-actin. Slides were mounted with Fluoromount G with Hoescht (Sigma) to label cell nuclei. The percentage of positive cells was determined for each assay with at least 300 cells being counted per condition using the counting feature on an Evos Xl core microscope. Each assay was run in triplicate and repeated three times. Representative images were taken on Zeiss Axio A1 Microscope with an AxioCam MRc digital camera.
3
Characterization of Nb-doped SrTiO3 Crystals
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Commercially available epi-polished SrTiO3:Nb (100) single crystals with doping concentration of 0.2, 1.0, 1.4 and 10.1 at% purchased from Crystec, Mateck, and SurfaceNet were investigated. Powder X-ray diffraction measurements were conducted on a STOE instrument using a Si reference in order to determine the lattice parameter precisely. AFM measurements were conducted on a JEOL instrument in tapping mode using a Si cantilever. Optical microscopy images were obtained by a Zeiss Axio A1 microscope in transmission light mode equipped with a 100 W LED light source adjusted to approx. 60%. Atom probe tomography was performed on needles cut out of the single crystals by FIB on a CAMECA LEAP4000 X HR instrument with UV-laser. TEM/EDX measurements were performed using an aberration corrected FEI Titan G2 80–200 transmission electron microscope equipped with a Super-X EDX detector. EDX maps have been recorded for 10 min with a lateral resolution of about 0.2 nm in 20–50 nm thick specimen areas. Simultaneously a HAADF image was acquired.
4
3D Single-Cell Colony Formation Assay
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Single cell suspension was seeded at a density of 100 cells/well in 96 well plates on the top of a thin layer of matrigel (BD Bioscience) [45 (link)]. RPMI cell culture media with 10% FBS and 4% matrigel was added on the top every other day. Cells were cultured up to 8 days. Colonies were counted and photographs were taken using a Zeiss Axio A1 microscope coupled with Zenoptik camera.
5
Histopathological Analysis of Mouse Tumor Infiltrates
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Abdominal walls and intestines from mice were fixed for at least 24 h in PBS-buffered 10% formalin. Tissues were routinely embedded in paraffin. 5 μm thick sections were stained routinely with H&E. For staining tumor-infiltrating T cells, mice were perfused with 4% paraformaldehyde (PFA) in PBS, and tumor nodules were fixed in 4% PFA/PBS for additional 2 hours, washed and infiltrated with 30% sucrose/PBS at 4°C. 6 μm thick frozen sections were stained with rat anti-mouse CD8 (BD Biosciences, 1:100 dilution) or rat anti-mouse Foxp3 (eBioscience, 1:12 dilution), followed by polyclonal rabbit anti-rat immunoglobulin/HRP (Dako, 1:750 dilution). Signal was developed with diaminobenzidine (DAB, Dako). Images were acquired on a Zeiss Axio A1 microscope. All histopathological and immunohistochemical samples were reviewed and the quantitation of the cellular infiltrate was performed in a blinded manner to the observer.
6
Wound Healing Assay: Gemcitabine and Digoxin
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Transfected or wild-type PANC-1 and MIA PaCa-2 cells were plated on 6-well plates until 90% confluent, and cell monolayers were scraped using a pipette tip. PBS was then used to remove cell debris. The monolayers were treated with gemcitabine (100 nM) and/or digoxin (40 nM) for 48 h, and the migrating cells were observed using an Axio A1 microscope (Carl Zeiss, Jena Germany). The wound healing area was then measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
7
Characterizing RXFP1 Knock-In Effects
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The effects of human RXFP1 knock-in were studied in late pregnant females at day 18.5 after vaginal plug detection as described previously [8 (link)]. Females were euthanized by CO2 overexposure and their reproductive organs measured, collected, and fixed in 4% paraformaldehyde. The length of the pubic symphysis was determined using a dissecting microscope equipped with an ocular micrometer [14 (link)]. Vaginal and pubic ligament sections at 6 to 7 μm were stained using Masson’s Trichrome Stain kit (Sigma-Aldrich Corp., St. Louis, MO) according to the manufacturer’s protocol. The thickness of vaginal epithelial cell layers was measured in 10 random cross sections for each sample of three to four females per group using AxioVision Software (Carl Zeiss Microscopy, Jena, Germany) on Carl Zeiss Axio A1 Microscope equipped with an AxioCam MRc5 CCD camera. The number of animals analyzed is indicated in figure legends. Previously published data for the wild-type, heterozygous +/Rxfp1-LacZ-, and Rxfp1-deficient females of the same mutant line as in the current experiments were used for comparisons [8 (link)]. Differences were expressed as mean ± standard error of the mean. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Bonferroni multiple comparison test using GraphPad software (La Jolla, CA).
8
Immunofluorescence Staining of Transfected Cells
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293T cells were plated on glass coverslips and transfected as described above. Cells were then fixed in 4% paraformaldehyde, blocked (3% BSA, 0.1% saponin, in PBS), and stained with primary antibodies diluted 1:250 in blocking buffer. Primary antibodies were M2 mouse anti-Flag (Sigma F3165-1MG) and rabbit anti-V5 (Cell Signaling Technology D3H8Q). The following secondary antibodies were used at a 1:1,000 dilution in blocking buffer: Alexa Fluor594 goat anti-mouse IgG (Life Technologies R37121) and Alexa Fluor488 goat anti-rabbit IgG (Life Technologies A27034). Cells were mounted in Vectashield medium containing 4,6-diamidino-2-phenylindole (Vector Laboratories) and imaged with a Zeiss Axio A1 microscope equipped with × 63/1.25 objective. Image acquisition was performed with the AxioVision (Rel.4.8) software package.
9
Kidney Fibrosis Quantification with Sirius Red
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Paraformaldehyde-fixed paraffin-embedded kidneys were sectioned at 4.5 μm thickness and stained with 0.1% sirius red solution (Electron Microscopy Sciences, Hatfield, PA, United States; Ng et al., 2017 (link)). At least 10 non-overlapping bright field images at 20× magnification were analyzed for each animal using the Carl Zeiss Axio A1 microscope with an AxioCam MRc5 CCD camera (Oberkochen, Germany). Staining was quantified using ImageJ software (NIH, Bethesda, MD; Schneider et al., 2012 (link)) and presented as percentage of stained area within the analyzed image.
10
Immunohistochemistry and TUNEL Assay of Testis
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Testis samples were fixed in 4% PFA overnight and embedded in paraffin. The embedded tissue was sectioned at 4.5 μm. H&E staining and IHC were performed as previously described [27 ]. Rabbit polyclonal antibodies to INPP4B (1:150 dilution, #8450, Cell Signaling, Danvers, MA) and γH2A.X (1:700, #2577, Cell Signaling, Danvers, MA) were used as primary antibodies and sections were counterstained with hematoxylin (Millipore). For TUNEL assay, ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Millipore) was used following manufacturer protocol. Three mice were analyzed for each group and minimum ten circular tubules were counted per mouse. The images were captured using a Carl Zeiss Axio A1 microscope with an AxioCam MRc5 CCD camera (Carl Zeiss, New York, NY).
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