Laser-capture microdissection was used to collect samples from the fascicles and IFM of SDFT samples from all age groups (n = 4 for each age group with the exception of the 0–1 month group where n = 3). For this purpose, 12 µm transverse cryosections were cut from the SDFT samples and mounted on steel frame membrane slides (1.4 µm PET membrane, Leica Microsystems, Wetzlar, Germany). Frozen sections were dehydrated in 70% and 100% ice-cold ethanol, allowed to briefly dry, and regions of fascicle and IFM laser-captured on an LMD7000 laser microdissection microscope (Leica Microsystems, Wetzlar, Germany) and collected in LC/MS grade water (FisherScientific, Hampton, New Hampshire). Collected samples were immediately snap frozen and stored at −80°C for mass spectrometry analysis.
Lmd7000 laser microdissection microscope
Manufactured by Leica
Sourced in Germany
The LMD7000 is a laser microdissection microscope designed for precise isolation of specific cells or regions from tissue samples. The instrument utilizes a laser beam to cut and capture targeted areas for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Lc ms grade water, by Thermo Fisher Scientific (1 mentions)
Mm99999915 g1, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Rnase free dnase 1, by Qiagen (1 mentions)
Rnalater solution, by Thermo Fisher Scientific (1 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Pen slides, by Leica (1 mentions)
4 protocols using lmd7000 laser microdissection microscope
1
Age-dependent Proteomic Analysis of SDFT
2
Isolation and Analysis of Intestinal Tumor Samples
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Freshly dissected intestinal tumors and normal intestinal tissues were immersed in RNAlater solution (Thermo Fisher Scientific) overnight and stored frozen at −80°C until further processing. The epithelial and stromal samples were prepared by microdissecting 7‐μm‐thick fresh frozen sections on polyethylene naphthalate membrane slides using an LMD7000 laser microdissection microscope (Leica Microsystems). Total RNA was isolated using an RNeasy Mini Kit (Qiagen) with on‐column RNase‐free DNase I (Qiagen) treatment. A High‐Capacity cDNA Reverse Transcription Kit (Life Technologies) was used for synthesis of complementary DNA from total RNA. DNA microarray analysis was carried out using an Agilent Technologies platform (Mouse GE 4 × 44K v2 Microarray). Real‐time PCR reactions were carried out using a 7500 Fast Real‐Time PCR system (Applied Biosystems, Life Technologies). The mRNA levels of Dio2, Vimentin, and Gapdh were quantified with the TaqMan Gene Expression assays (Mm00515664_m1, Mm01333430_m1, and Mm99999915_g1, respectively) from Applied Biosystems. Gapdh was used as an internal control to normalize the expression levels of Dio2 and Vimentin by the comparative 2−ΔΔCT method.20
3
Laser-Capture Microdissection for DNA Extraction and Analysis
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Laser-capture-microdissected tissues were cut to 15 μm thickness, mounted on PEN slides (Leica Microsystems), and air-dried at room temperature for 1 hr. Slides were stored in sealed slide mailers at −80°C until use. Microdissection was carried out on a Leica Microsystems LMD7000 laser microdissection microscope, sorting a single colonic crypt section or five smooth muscle fibers into single tubes for further analysis. Captured colonic crypts or muscle fiber sections were settled to the bottom of the tube by centrifugation at 7,000 rcf for 10 min. DNA was extracted in 10 μl lysis buffer (50 mM Tris-HCl [pH 8.5], 1% Tween-20, and 20 mg/ml proteinase K) for at least 2 hr and 55°C, with a heat-inactivation step at 95°C for 10 min (Taylor et al., 2003 (link)). The extraction was used directly in PCR reactions for mutation level quantification or diluted in 30 μl water for PCR and sequencing.
4
Laser Microdissection of Epithelial Cells
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We used an LMD7000 Laser Microdissection Microscope (Leica Microsystems, Wetzlar, Germany) with 10× magnification and proper laser settings to microdissect the mounted and stained tissues from the previous section. Only epithelial layers with a target size of 0.06 mm2, which corresponded to about 600 cells, were microdissected. We placed each 600-cell microdissected isolate into an empty cap of a nuclease-free 0.2 ml Axygen PCR tube (Thermo Fisher Scientific). We took photomicrographs both before and after LCM.
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