Cells were treated with 20 ng ml–1 KaryoMAX colcemid solution (Thermo Fisher Scientific, 15212012) for 2 h at 37 °C in a humidified incubator. Cells were collected in 0.8% sodium citrate solution (Sigma-Aldrich, S4641) and maintained at 37 °C for 30 min. The cell suspension was fixed with 3:1 methanol:acetic acid (Chemie Brunschwig, M/4000/17; FSHA/0406/PB08) added drop-by-drop, washed twice in the fixative solution and incubated overnight at −20 °C. Cells were dropped onto a glass slide (Thermo Fisher Scientific, J1800AMNZ). Slides were incubated for 2 min in a humidified chamber at 65 °C and air-dried at room temperature for 30 min. Slides were mounted and DAPI-stained concomitantly with ProLong Diamond antifade mountant with DAPI (Thermo Fisher Scientific,. P36962) according to the manufacturer’s instructions. Metaphases were imaged at ×100 resolution on a Zeiss Axioplan upright microscope. Images were analysed using Fiji (v.2.9.0)53 (link).
Axioplan upright microscope
Manufactured by Zeiss
Sourced in Germany
The Axioplan upright microscope is a high-performance laboratory instrument designed for a variety of microscopy applications. It features a robust and ergonomic design, providing a stable platform for precise optical observations.
Automatically generated - may contain errors
Lab products found in correlation
J1800amnz, by Thermo Fisher Scientific (1 mentions)
S4641, by Merck Group (1 mentions)
Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Karyomax colcemid solution, by Thermo Fisher Scientific (1 mentions)
Ab4448, by Merck Group (1 mentions)
A7906, by Merck Group (1 mentions)
T6074, by Merck Group (1 mentions)
P7280, by Merck Group (1 mentions)
Alexa fluor 488 anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Anti mouse igg alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
10 protocols using axioplan upright microscope
1
Metaphase Chromosome Preparation and Imaging
2
Immunofluorescence Staining of Cells
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Cells were cultured on coverslips coated with poly-d -lysine (Sigma-Aldrich, P7280) and incubated in standard conditions. Cells on coverslips were fixed with ice-cold methanol at 4 °C for 30 min. Cells were washed with PBS multiple times and incubated with 5% BSA (Sigma-Aldrich, A7906) at room temperature for 30 min. Next coverslips were incubated with primary antibodies at the indicated concentrations against pericentrin (0.1 μg ml–1; Abcam, ab4448) and α-tubulin (0.5 μg ml–1; Sigma-Aldrich, T6074) diluted in 1% BSA for 1 h at room temperature in a humidified chamber. Coverslips were washed with PBS and incubated with the fluorescent secondary antibodies anti-mouse IgG-Alexa Fluor 594 (2 μg ml–1; Thermo Fisher Scientific, A-11005) and anti-rabbit IgG-Alexa Fluor 488 (2 μg ml–1; Thermo Fisher Scientific, A-11034) diluted in 1% BSA for 1 h at room temperature in the dark. Coverslips were washed in PBS followed by mounting and counterstaining with DAPI with ProLong Diamond antifade mountant. Cell images were captured at ×63 resolution on a Zeiss Axioplan upright microscope. Images were analysed using Fiji (v.2.9.0)53 (link).
3
Immunohistochemical Analysis of p53 in Eye
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Histochemical analysis was performed on paraformaldehyde-fixed (PFA 4%), paraffin-embedded 2 μm thick cross sections of eye. Slices were washed in PBS and blocked in PBS containing 10% goat serum and 0.5% of triton X-100. Immunohistochemistry was performed using a rabbit polyclonalanti-p53 (1:100, Santa Cruz Biotechnology; cat no: sc-6243; Germany). As secondary antibody, an anti-rabbit-biotinylated antibody was used (Jackson Immuno Research Europe; UK). Microscopic images of stained eye-section were taken with an Axioplan upright microscope (Zeiss; Germany) using AxioVision 4.8.2 software. Exposure time was kept constant for all investigations of p53-staining in all eye-sections.
4
High-Speed Videomicroscopy of Colloidal Gold-Labeled Molecules
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For the observations with enhanced frame rates, a digital high-speed camera with a C-MOS sensor was used (FASTCAM-ultima; Photron, Tokyo, Japan; Tomishige et al., 1998 (link); Fujiwara et al., 2002 (link)). For high-speed videomicroscopy of colloidal gold–labeled molecules, bright-field optical microscopy was used, using a Zeiss Axioplan upright microscope equipped with an αPlan-Fluar 100× oil immersion objective lens (NA 1.45). The sequence of images was replayed at the video rate (30 Hz) with analogue and digital enhancement by an image processor (DVS-3000; Hamamatsu Photonics, Hamamatsu, Japan) and recorded on a digital videotape recorder (DSR-20; Sony, Tokyo, Japan).
The precision of the position determination was estimated by the same method used in SFMT using 40-nm-diameter gold particles and was 17 nm at a time resolution of 25 μs (Fujiwara et al., 2002 (link)).
The precision of the position determination was estimated by the same method used in SFMT using 40-nm-diameter gold particles and was 17 nm at a time resolution of 25 μs (Fujiwara et al., 2002 (link)).
5
Whole-Mount Alcian Blue and Alizarin Red Staining Protocol for Zebrafish Larvae
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The larvae were euthanized with tricaine and then fixed in 2% PFA at room temperature for 1 h. Then, the larvae were washed with 100 mM Tris (pH 7.5) and 10 mM MgCl2 for 10 min (min) and incubated overnight in Alcian stain (stock: 0.1 g Alcian blue 8GX, 2.6 ml H2O, bring the volume to 50 ml by using 95% EtOH; working solution: 10 ml Alcian blue stock, 32.6 ml 95% EtOH, 5 ml Tris-HCl, pH 7.5, 0.5 ml 1M MgCl2, 0.9 ml H2O) at room temperature. The fish were destained by 80% EtOH/100 mM Tris (pH 7.5)/10 mM MgCl2, 50% EtOH/100 mM Tris (pH 7.5)/10 mM MgCl2, and 25% EtOH/100 mM Tris (pH 7.5)/10 mM MgCl2 for 5 min each. The fish were moved into a well of 24-well plates and bleached by 3% H2O2/0.5% KOH under a light source until the eyes turn to light brown. The fish were washed twice with 1 ml 25% glycerol/0.1% KOH for 10 min each and nutated in alizarin stain (stock: 0.25 g alizarin red S to 50 ml H2O, working solution: 1 ml alizarin red stock, 12.5 ml glycerol, 5 ml Tris-HCl, pH 7.5, 31.5 ml H2O) at room temperature for 1 h. The fish were destained by washing with 1 ml 50% glycerol/0.1% KOH for 10 min twice. Then gradually move them into 100 % glycerol and image the fish in glycerol using a Zeiss Axioplan Upright Microscope in a bright field.
6
Seed Viability Staining Protocol
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Seeds were stained with 1% solution of 2,3,5-triphenyltetrazolium chloride (Sigma) and incubated for 8 h in root temperature. The tetrazolium salts were converted into highly colored end products known as formazans through a process of metabolic reduction. Photographs of the stained seeds were taken using a Zeiss Axioplan upright microscope.
7
Quantitative Analysis of GFP Expression
8
Embryo Fixation and Paraffin Embedding
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Embryos were harvested and fixed O/N at 4 °C in 0.1 m phosphate buffer (PB) containing 4% (w/v) paraformaldehyde (PFA). Fixation time varied from O/N (minimum) for E10.5–E12.5 embryos to longer times as appropriate for larger embryos. Embryonic tissues were dehydrated in serial alcohols before clearing in a proprietary clearing agent (Histoclear‐National Diagnostics) followed by paraffin wax embedding. After sectioning, paraffin‐embedded sections were dewaxed, re‐hydrated, stained with hematoxylin before being counterstained with eosin. Images were obtained using a Zeiss AxioPlan upright microscope.
9
Hematoxylin and Eosin Tissue Staining
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Tissues were cryosectioned to 12µm thickness and subjected to Hematoxylin and Eosin staining. Briefly, sections were air dried for 15 min and dipped in isopropanol solution for 10 min. The sections were then washed in distilled water and stained with Harris hematoxylin solution for 2 min in a dark place. Following washing with distilled water, slides were stained with eosin and immediately washed in distilled water. The slides were further dehydrated by treating with 50%, 70%, 95% and 100% ethanol. Finally, the slides were cleared in xylene and mounted using DPX mountant and visualized using Axioplan upright microscope (Zeiss).
10
Amyloid Plaque Visualization in Mouse Brain
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The coronal brain sections of mice corresponding to various treatment groups were subjected to Congo red staining to visualize amyloid plaques. The protocol has been described in detail elsewhere 18 . Briefly, the slide mounted, overnight air-dried brain cryosections were hydrated, followed by treatment with 80% ethanol saturated with NaCl for 20 min. The slides were subsequently treated with 0.2% Congo red solution prepared in 80% ethanol saturated NaCl solution. Prior to the treatment, both solutions were rendered alkaline by the addition of 10 mM NaOH. After 30 min exposure to Congo red solution, slides were washed in 95% and 100% ethanol and cleared in xylene. The coverslips were mounted using DPX mountant, and imaged using Axioplan upright microscope (Zeiss). Congo red-positive amyloid plaques were quantitated using NIH-ImageJ software. The images were segmented by HSB method to differentiate background and the Congo red positive pixels, and the area of these pixels were quantified.
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