Homozygous Venus::HES5 knock-in females were mated with R26R-H2B::mCherry Sox1Cre:ERT2 males and E0.5 was considered as midday on the day a plug was detected. Intra-peritoneal injection of pregnant females with 2.5 mg Tamoxifen (Sigma) was performed 18 h prior to embryo dissection. Whole embryos were screened for H2B::mCherry expression using Fluar ×10/0.5NA objective on a Zeiss LSM880 confocal microscope and the trunks of positive embryos were embedded in 4% low-gelling temperature agarose (Sigma) containing 5 mg/ml glucose (Sigma). 200 μm transverse slices of the trunk around the forelimb region were obtained with the Leica VT1000S vibratome and released from the agarose. Embryo and slice manipulation was performed in phenol-red-free L-15 media (Thermo Fisher Scientific) on ice and the vibratome slicing was performed in chilled 1xPBS (Thermo Fisher Scientific).
Agarose
Manufactured by Leica
Agarose is a polysaccharide derived from red algae, commonly used as a gel matrix in various laboratory applications, such as gel electrophoresis and chromatography. It provides a stable, inert, and porous structure that allows the separation and analysis of biomolecules like DNA, RNA, and proteins.
Automatically generated - may contain errors
Lab products found in correlation
Vt1000, by Leica (2 mentions)
Vt1000s vibratome, by Leica (1 mentions)
L 15 media, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Tamoxifen, by Merck Group (1 mentions)
Lsm 880 confocal microscope, by Zeiss (1 mentions)
Low gelling temperature agarose, by Merck Group (1 mentions)
Agarose, by Lonza (1 mentions)
Vt1200, by Leica (1 mentions)
Seaplaque agarose, by Lonza (1 mentions)
Calcofluor white m2r, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Agarose, by Thermo Fisher Scientific (1 mentions)
Rabbit anti gfp antibody, by Merck Group (1 mentions)
Isoflurane, by Baxter (1 mentions)
4 protocols using agarose
1
Tamoxifen-Induced Lineage Tracing
2
Retinal tissue sectioning and preservation
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Retinal punches from the macula and peripheral regions of donor eyes were fixed in 4% paraformaldehyde (PFA) in PBS for 2 hr and then transferred to PBS for 1 hr. Retinal pieces were embedded in low melting point agarose (42°C, 3% agarose (Lonza) in PBS). Tissue blocks were cooled down at 4°C until the agarose solidified. agarose blocks were trimmed and glued to the metal chuck of a vibratome (Leica, VT1200S). Vibratome sections (100 μm thick) were obtained from retinal tissues and stored in PBS at 4°C.
3
Arabidopsis Xylem Pattern Phenotyping
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For Arabidopsis xylem pattern phenotyping, 5 DAT (days after transfer to growth chamber from stratification) seedlings were used. Five to six seedlings overlaid straight on a MS plate were pulled together and then dipped into 4% low-melting temperature SeaPlaque®, agarose (Lonza), which was melted in 1 × PBS buffer (pH 7.5). Next, the seedlings in the 4% agarose solution were placed in disposable base molds (30 mm × 24 mm × 5 mm). The solidified agarose was cut into a block and sectioned using a vibratome (Leica VT1000S), resulting in thicknesses in the range of 100–120 μm. For observation of the cell boundaries under a confocal microscope, each slice was stained with 10 μg/ml of a Calcofluor white M2R (Sigma-Aldrich) solution.
4
Perfusion and Immunostaining of Mouse Brains
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Mice were anesthetized with isoflurane (Baxter Healthcare Corporation) and perfused intracardially with PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4/ 2H2O, 1.4 mM KH2PO4; all from Sigma-Aldrich) and 4% paraformaldehyde (PFA, Merck) in PBS prior to decapitation. Brains were removed and after post-fixation in ice-cold 4% PFA for 2 h each brain was embedded in 2.5% agarose (Invitrogen)/PBS. agarose embedded brains were sliced (70 μm sections) on a vibratome (VT1000s, Leica). To increase the contrast of the Venus signal in the thick vibratome sections, the Venus fluorescence was visualized together with its immunoreactivity by using a rabbit anti-GFP antibody (1:5,000, Millipore) and FITC-coupled anti-rabbit secondary antibody (1:200, Jackson ImmunoResearch). For the NeuN staining, mouse anti-NeuN antibody were used (1:1,000, Merck Millipore) and followed with Cy3-coupled anti-mouse secondary antibody (1:200, Jackson ImmunoResearch). Immunstaining was performed as described (Krestel et al., 2001 (link)).
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