The A549 (3,000 cells) or H1299 (3,000 cells) cells were seeded on silane coated micro slides (Muto pure chemicals Co.) with the same cell density and transfection concentration as MTT assay. The cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) at 48 h following transfection. The cell membrane was damaged by 0.1% Triton X-100 (Mallinckrodt Specialty Chemicals Co) and blocked with 2% bovine serum albumin (Sigma-Aldrich). Ki-67 primary antibody (1:400, #9129, Cell Signaling Technology) and anti-rabbit IgG secondary antibody (1:800, #8889, Cell Signaling Technology) were used for Ki-67 detection at a wavelength of 550/580 nm. The incubation conditions for the primary antibody were 4°C overnight, and for the secondary antibody they were 2 h at room temperature. Cell nuclei were stained with Hoechst 33342 (#B2261, 1 µg/ml, Sigma-Aldrich) and detected at wavelength of 360/460 nm. The incubation conditions for Hoechst 33342 were 30 min at room temperature. Images were acquired using the Zeiss AxioImager A1 system (Carl Zeiss).
Axioimager a1 system
Manufactured by Zeiss
Sourced in Germany
The Zeiss AxioImager A1 system is a microscope designed for brightfield, phase contrast, and fluorescence imaging. It features a motorized nosepiece, objectives, and stage for precise sample positioning and observation. The AxioImager A1 system is equipped with a high-resolution camera and specialized software for image acquisition and analysis.
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Lab products found in correlation
Ki67 primary antibody, by Cell Signaling Technology (1 mentions)
Anti rabbit igg secondary antibody, by Cell Signaling Technology (1 mentions)
Silane coated micro slides, by Muto Pure Chemicals (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Rotary microtome, by Leica (1 mentions)
Poly lysine coated chamber slides, by BD (1 mentions)
Quantigene viewrna assay kit, by Thermo Fisher Scientific (1 mentions)
Axiovision software, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Southern Biotech (1 mentions)
4 protocols using axioimager a1 system
1
Ki-67 Immunofluorescence Staining Protocol
2
Microscopic Analysis of S1 Stage Seeds
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Six S1 stage seeds were collected and fixed for 12 h at 4 °C in 2% glutaraldehyde in 0.02 M phosphate buffer at pH 7.0. Samples were dehydrated in an ethanol series and acetone, and embedded in Spurr’s resin (SPI-CHEM). Sections 5µm thick were cut with a rotary microtome (Leica) and stained with Toluidine blue O for examination under a Zeiss microscope. Images were photographed using the associated Zeiss Axio Imager A1 system.
3
RNA-FISH Analysis of Rubella Virus
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Cells grown on poly-lysine coated chamber slides (BD Biosciences) were infected with RV at an MOI of 5 and then treated with 2.5 μg/ml NTZ for 2 days. RV RNA-FISH using a QuantiGene ViewRNA assay kit (Affymetrix) has been described (Perelygina et al., 2015 ). A mixture of a Cy5-labeled probe set for either negative or positive-strand RV genomic RNA or influenza negative strand (negative control) and a FITC-labeled probe set for the peptidylpropyl isomerase B (PPIB) or β-actin gene was used. Images were taken using an AxioImager A1 system and AxioVision software (Zeiss, Germany). Fluorescence intensity from six microscopic fields (40–50 cells per field) was measured using ImageJ (Schindelin et al., 2012 (link)).
4
Visualization of Neutrophil Extracellular Traps
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After fixation with PFA, NETs were washed (three times) with PBS, blocked with 1% BSA in PBS over night at 4°C, and incubated first with 20 µg/mL anti-human ZPI antibody (Molecular Innovation) in PBS for 30 min at room temperature and then with 2 µg/mL Alexa Fluor 594 donkey anti-sheep IgG (ThermoFisher Scientific) in PBS for 30 min at room temperature. The slides were mounted in medium containing 4',6diamidino-2-phenyl indole (DAPI, SouthernBiotech, Birmingham, AL, USA) to label DNA, and widefield microscopy images were acquired on an AxioImager A1 system (Carl Zeiss, OberKocher, Germany).
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