To prepare thin slices for imaging, biofilm colonies were grown as follows: cells were scraped from an overnight LB plate and re-suspended in LB medium to A600 = 1.0 optical units. 2 μL of cell suspension was put on the surface of an MSgg agar plate. Biofilm colonies were collected at 24, 36, 48, 72, and 96 h after incubation at 30 °C and flash-frozen in liquid nitrogen overlaid with Tissue-Tec O.C.T. compound (Sakura Finetek, ref 4583) in custom foil moulds and kept at −80 °C. 8 μm thin slices were made using a Leica CM3050S cryomicrotome, collected on SuperFrost Plus microscope slides (VWR cat. no. 631-0108) and stored at −80 °C before use. For imaging, biofilm slices were overlaid with the mounting medium (0.5% N-propyl gallate, 50% glycerol and PBS (pH 7.4))24 (link).
Cm3050s cryomicrotome
Manufactured by Avantor
The CM3050S cryomicrotome is a laboratory instrument used for the preparation of thin samples for microscopic analysis. It is designed to section frozen biological samples at low temperatures, enabling the preservation of their structural integrity. The cryomicrotome provides precise control over the cutting thickness and temperature to ensure high-quality sample sections.
Automatically generated - may contain errors
Lab products found in correlation
Superfrost plus micro slide, by Avantor (3 mentions)
X gal, by Avantor (2 mentions)
Superfrost plus microscope slides, by Avantor (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Ckx41 microscope imaging system, by Olympus (1 mentions)
Senescence β galactosidase staining kit, by Beyotime (1 mentions)
4 protocols using cm3050s cryomicrotome
1
Preparation of Biofilm Samples for Cryogenic Imaging
2
Senescence-Associated β-Galactosidase Staining
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SA-β-Gal staining was performed following the previously published protocols (Debacq-Chainiaux et al., 2009 (link); Ma et al., 2020 (link), 2021 (link); Wang et al., 2018 (link)). In brief, the O.C.T-embedded ovarian tissues were cryosectioned at a thickness of 8 μm with a Leica CM3050S cryomicrotome, mounted on Superfrost Plus microslides (VWR) and stored at −80°C. Before SA-β-Gal staining, sections were dried at room temperature (RT), fixed in fixation buffer (2% formaldehyde and 0.2% glutaraldehyde) for 5 min and stained with freshly prepared SA-β-Gal staining solution (1 mg/mL X-gal (Amresco, 0428), 5 mmol/L K4[Fe(CN)6], 5 mmol/L K3[Fe(CN)6], 2 mmol/L MgCl2, 150 mmol/L NaCl, 40 mmol/L citric acid/Na phosphate buffer) at 37°C for 2 weeks. Further, the sections were counterstained with Nuclear Fast Red Staining Solution (Beyotime, C0151) to visualize the nucleus. Finally, the sections were dehydrated and sealed with resinous mounting medium. Images were taken with Olympus CKX41 microscope imaging system, and the SA-β-Gal-positive areas were quantified with ImageJ.
3
Senescence-Associated β-Galactosidase Staining of Mouse Kidney
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SA-β-Gal staining was performed using a previously published protocol (Debacq-Chainiaux et al., 2009 (link); Geng et al., 2019 (link); Zhang et al., 2021 (link)). In brief, OCT-embedded, snap-frozen, unfixed mouse kidney tissues were cryosectioned at a thickness of 15 μm with a Leica CM3050S cryomicrotome, collected on Superfrost Plus microslides (VWR) and kept at –80°C until use. For SA-β-Gal staining, sections were thawed at RT and processed by using Senescence β-Galactosidase Staining Kit (Beyotime, Cat#C0602) as the manufacturer’s recommendation. Images were taken with Carl Zeiss 200 microscope, and the SA-β-Gal-positive areas were quantified using ImageJ.
4
Senescence-Associated β-Galactosidase Staining
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SA-β-Gal staining was performed using a previously published protocol (Debacq-Chainiaux et al., 2009 (link); Chow et al., 2019 (link); Ma et al., 2020a , 2020b (link)). In brief, the hippocampal tissue stored in the cryotube was fixed and dehydrated in a cold mixture of 75% ethanol and 4% paraformaldehyde at 4 °C shaking for 3 h, then in 4% paraformaldehyde at 4 °C shaking overnight, followed by shaking in 5% sucrose at 4 °C for 3 h and then in 30% sucrose at 4 °C shaking overnight or until the tissue shrinked completely. OCT-embedded, dehydrated hippocampus tissues were cryosectioned at a thickness of 10 μm with a Leica CM3050S cryomicrotome, collected on Superfrost Plus microslides (VWR) and stored at −80 °C until use. For SA-β-Gal staining, sections were thawed at RT and rinsed in PBS, fixed in 2% formaldehyde and 0.2% glutaraldehyde at RT for 5 min and stained with freshly prepared staining solution at 37 °C (X-gal was purchased from Amresco; all the other reagents were from Sigma-Aldrich). Images were taken with PerkinElmer Vectra Polaris, and the SA-β-Gal-positive areas were quantified using ImageJ.
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