For LMD of human samples, a PALM Zeiss Microbeam 4 was used. Sample TMA blocks were cut into 10‐μm‐thick sections and mounted on superfrost slides. For LMD of mouse samples, a Leica LMD6000 was used. Tissue blocks were cut into 10‐μm‐thick sections and mounted on membrane slides (PEN Membrane, 2.0 μm, Leica). LMD was conducted similarly for mouse and human samples: For each sample, a slide was stained with hematoxylin and eosin for inspection before LMD. To obtain the minimum amount of tissue (100 nl = 0.1 mm³) necessary for consecutive LC‐MS/MS analysis, at least 10 mm² of target area were laser‐microdissected. To obtain proteomic profiles solely from the tumor, stroma and immune cells were excluded from dissection. In tumor samples, only cancerous prostate glands were dissected. Microdissected FFPE samples were stored at −20°C before LC‐MS/MS analysis.
Pen membrane
Manufactured by Leica camera
Sourced in Germany
The PEN Membrane is a highly specialized laboratory equipment designed for precise filtration and separation applications. It features a durable polyethylene membrane that provides efficient performance and reliability in various research and analytical settings. The core function of the PEN Membrane is to facilitate the filtration and separation of samples, ensuring precise and consistent results.
Automatically generated - may contain errors
Lab products found in correlation
Lmd6000, by Leica camera (2 mentions)
Plus slides, by Thermo Fisher Scientific (1 mentions)
Lmd7000, by Leica camera (1 mentions)
Leica cm1950, by Leica camera (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Bioanalyzer rna pico kit, by Agilent Technologies (1 mentions)
Smarter stranded total rna sample prep kit, by Takara Bio (1 mentions)
Rnaeasy ffpe kit, by Qiagen (1 mentions)
Nextseq 500, by Illumina (1 mentions)
5 protocols using pen membrane
1
Laser Microdissection and Proteomic Analysis of Prostate Tissue
2
Formalin-Fixed Paraffin-Embedded Tissue Preparation
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Archival or discarded surgical tissue was obtained from the Johns Hopkins Pathology Department under IRB exemption IRB00089413. Tissues were fixed in formalin for at least 48 h prior to dehydration and paraffin wax embedding. 10 μm thick sections were prepared using RNase-free precautions for standard (Plus slides; Thermo Fisher, Waltham, MA) and laser capture microdissection (LCM) (Leica PEN-membrane) slides. FFPE blocks and sections were stored in desiccant; blocks were stored at room temperature and sections were stored at −20°C.
3
Glomerular Isolation and Immunoblotting
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Murine kidneys were frozen in O.C.T. Compound (Sakura Finetek Japan, Tokyo, Japan). The sections (10 µm-thick) were prepared using Leica CM 1950 (Leica, Wetzlar, Germany) and attached to PEN Membrane (Leica). After fixing with ethanol, the nuclei were stained with hematoxylin. Approximately 350 glomeruli for each sample were isolated and collected using LMD7000 (Leica). The samples were then subjected to immunoblotting.
4
Tissue Sampling and RNA Sequencing Protocol
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Fresh-frozen tissue samples were partially thawed in 1% formaldehyde/PBS (Phosphate-buffered saline) and continued to fix for 10 min at room temperature. Fixed tissue was rinsed in PBS and embedded in OCT (optimal cutting temperature) compound (4583 Tissue-Tek, Sakura Finetek, Tokyo, Japan). Tissue samples were sectioned (10 μm) and placed on membrane slides (Pen-membrane, Leica, Tokyo Japan). Sections were stained with hematoxylin and dehydrated by ethanol. Tissue parts were obtained by LMD (LMD6000, Leica) [12 (link)]. For the isolation of total RNA, sections were processed with an RNAeasy FFPE Kit (73504, Qiagen, Hilden, Germany). RNA yield and degradation status were monitored by a Bioanalyzer RNA Pico Kit (5067–1513, Agilent Technologies, Santa Clara, CA, USA). Total RNA samples (quantities in ng; Normal: range = 10.5–64.7, mean = 33.6; Tumor: range = 2.2–98.2, mean = 42.7; Stroma: range = 4.2–110, mean = 28.5) were converted into sequencing libraries using a SMARTer Stranded Total RNA Sample Prep Kit (635005, Takara Bio, Shiga, Japan). Sequencing was performed with Illumina NextSeq500 (Illumina, San Diego, CA, USA) to obtain 2 × 36-base reads.
5
Laser Capture Microdissection of Mouse Retina
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Eyes were enucleated from normal and diabetic mice and frozen at −80°C in the optimal cutting temperature (OCT) compound. Also, 20-μm thick cryosections were cut and placed on frame slides polyethylene naphthalate (PEN)-membrane (Leica, Wetzlar, Germany) for LCM. Slides were placed in 95% ethanol for 1 minute with gentle inversion up and down, followed by 75% and 50% ethanol for 30 seconds each. Sections were then stained with cresyl violet for 40 seconds and dehydrated in 50%, 75%, 95%, and 100% ethanol, respectively, for 30 seconds each. Sections were placed in xylene for 5 minutes and then placed at room temperature to dry. A Leica LMD 7000 LCM machine was used to isolate the outer retina (photoreceptor region; i.e., outer nuclear layer [ONL], inner segment/outer segment [IS/OS]) from the remaining inner retina (i.e., outer plexiform layer [OPL], inner nuclear layer [INL], inner plexiform layer [IPL], and ganglion cell layer [GCL]; Supplementary Fig. S1 ). Isolated regions were placed in lysis buffer, and RNA was isolated using the Qiagen RNeasy Micro Kit (cat no. 74004; Qiagen, Hilden, Germany).
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