1 × 105 cells were seeded into coverslips and incubated for 24 h. The cells were then treated with anoplin (64 μM) or its derivatives (8–32 μM) for 24 h. Next, cells were washed with PBS and fixed in 4% PFA for 15 min at room temperature. After three washes with PBS, cells were stained with DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride; Boster) for 5 min (as indicated in the instruction). Coverslips with cells were washed three times and mounted on a glass microscope slide using a Dako Fluorescence Mounting Medium. Images were acquired with Axio Imager Z2 LSM 700 Zeiss confocal microscope.
Axio imager z2 lsm 700 confocal microscope
Manufactured by Zeiss
Sourced in Germany
The Axio Imager Z2 LSM 700 is a confocal microscope manufactured by Zeiss. It is designed for high-resolution, three-dimensional imaging of biological samples. The microscope features a laser scanning system and advanced optics to provide detailed, high-quality images.
Automatically generated - may contain errors
Lab products found in correlation
Superfrost plus slide, by Thermo Fisher Scientific (2 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Boster Bio (1 mentions)
Alexa fluor 594 conjugate, by Thermo Fisher Scientific (1 mentions)
Ab6121, by Merck Group (1 mentions)
Mabn50, by Merck Group (1 mentions)
Ab9610, by Merck Group (1 mentions)
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (1 mentions)
Cm1860 cryostat, by Leica (1 mentions)
Prolong gold containing dapi, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
4 protocols using axio imager z2 lsm 700 confocal microscope
1
Anoplin and Derivatives Microscopy
2
Immunofluorescence Staining of OPCs and Brain Slices
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OPCs were fixed, washed with PBS, blocked in 3% donkey serum in PBST, and then incubated overnight with primary antibodies (anti‐PDGFR alpha, Abcam, ab6121; anti‐OLIG1, Merck Millipore, MAB5540, anti‐MBP, ProteinTech, 10458‐1‐AP) at 4°C. Free‐floating brain slices were washed with PBST, blocked in 5% donkey serum in PBST, and incubated overnight with primary antibodies (mouse anti‐OLIG2, 1:250, Merck Millipore, catalog number MABN50; rabbit anti‐OLIG2, Merck Millipore, catalog number AB9610; anti‐TCF7l2, 1:250, Merck Millipore, catalog number 05‐511; anti‐PDGFR alpha, Abcam, ab6121; anti‐OLIG1, Merck Millipore, MAB5540) at 4°C. On the next day, OPCs and brain slices were washing and they were incubated with Alexa Fluor‐conjugated secondary antibodies (anti‐Rabbit IgG (H + L), Alexa Fluor® 488 conjugate, ThermoFisher Scientific, A‐21206; anti‐Mouse IgG (H + L) Secondary Antibody, Alexa Fluor® 594 conjugate, ThermoFisher Scientific, A‐21203). Cell nuclei were counterstained with DAPI (OPCs) or Hoechst (brain slices). The images were captured with Nikon Eclipse Ni‐U fluorescence microscope or Axio Imager Z2 LSM 700 Zeiss Confocal Microscope.
3
Cryosectioning and Immunofluorescence of Zebrafish Larvae
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Zebrafish larvae were fixed overnight at 4°C in 4% PFA/PBS. The next day, samples were cryoprotected with 30% sucrose and later embedded in Optimal Cutting Temperature (OCT) medium. Frozen samples were sectioned in a Leica CM1860 cryostat (Leica Microsystems, Wetzlar, Germany) into 20 μm thick slices mounted on glass SuperFrost Plus slides (Thermo Fisher Scientific, Waltham, United States). The slides were stored at –20°C. On the day of staining, the sections were brought to RT, washed three times in PBS and once in PBS-Tween. Then the sections were incubated with 1% bovine serum albumin and 2% normal donkey serum in PBS-Tween overnight. The next day, the sections were washed in PBS and primary antibodies (Supplementary Table 3 ) were applied overnight at 4°C. Next, slides were washed in PBS-Tween and incubated with secondary antibodies for 1 h at RT. Finally, slides were washed in PBS and sealed with coverslips in Fluoromount with DAPI to stain cell nuclei. The stained slides were stored at –20°C or visualized under a Zeiss Axio Imager Z2 LSM700 confocal microscope.
4
Immunofluorescence Staining of Bone Sections
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Long bones were fixed for 48 hr in 10% formalin, decalcified in 0.5 M EDTA (pH 7.5) for 3 days at 4 °C, dehydrated in 30% sucrose in PBS overnight and embedded in OCT. Antigen retrieval of 8‐µm sections was performed in citrate buffer 10 mM pH 6, 0.5% Tween20 (2 hr) followed by blocking with 3% BSA/0.2% Triton in PBS. Cultured cells were fixed with 4% formaldehyde in PBS for 15 min at RT, permeabilized in cold 100% methanol for 2 min at −20 °C, washed twice with PBS and blocked with 0.01% Tween/1% BSA in PBS. Slides with bone sections or cells were stained with rabbit‐anti‐mTNW11 and the mouse monoclonal anti‐hTNW56O15 followed by secondary antibodies Alexa Fluor 568 (Invitrogen, Life Technlogies Europe, Zug, Switzerland). Slides were mounted with ProLong Gold containing DAPI (Invitrogen) and images acquired using an Axio Imager Z2 LSM700 confocal microscope (Zeiss, Jena, Germany).
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