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Because convergence in the brain occurs in three dimensions, our first approach was to collect sections from a 50 µm×50 µm area at a depth of 9 µm and merge them into a single image.
Tissue samples were first subjected to cryosectioning to collect sections for hematoxylin and eosin (H&E) staining as well as for the isolation of RNA.
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Additionally, the first 10 20 sections cut from a specimen were discarded prior to collecting sections for DNA extraction from the specimen.
In an attempt to collect only sections from the distal side of the lamina, semithin sections were cut until the first cartridges within the lamina neuropil could be seen after which ultrathin 65 nm sections were made.
A cryostat was used to collect sagittal sections of 14-μm thickness.
A Leica cryostat (model Jung CM 3000, Wetzlar, Germany) was used to collect coronal sections (15 μm).
To study the clinical significance, we performed LCM to collect cancer sections from tumour tissues, qRT PCR, and immunohistochemistry in 24 patients who underwent R0 resection.
Cryoprotectioning, tissue embedding and serial cryosectioning were performed as described previously using a Leica cryostat (CM1850, 160 mm steel blade with C profile) at −24°C and SuperFrost Plus microscope slides to collect the sections.
Samples were collected either by submerging sample bottles directly into the center of the channel for wadeable streams or by using a weighted-bottle sampler to collect cross-section integrated samples from a bridge for nonwadeable streams (11).
When requesting FFPE sections, the best practice is to collect two additional sections (immediately before and after the section(s) being tested) that can be used for H&E staining to assess the tumor content and pathology of the sample analyzed.
Collected Sections were homogenized in TRIzol (Invitrogene, Carlsbad, CA) by using an Ultra-Turrax homogenizer (Janke and Kunkel, Staufen, Germany).
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