Pen membrane slides

Manufactured by Thermo Fisher Scientific

Sourced in United States

PEN membrane slides are a specialized type of microscope slide designed for laser capture microdissection (LCM) applications. These slides feature a thin, transparent polyethylene naphthalate (PEN) membrane that allows for the precise isolation of targeted cells or tissue samples from a larger sample. The membrane provides a clean surface for microdissection, enabling researchers to extract specific cells or regions of interest for further analysis.

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PEN Membrane Frame Slides PEN membrane glass slides PEN membrane covered slides LCM PEN membrane slide

19 protocols using pen membrane slides

Motor Neuron Retrograde Labeling

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Nine FVB mice from the same wild-type FVB litter at p14 were divided into three groups according to the muscle location being injected, resulting in groups of 3 mice per muscle. Mice were anaesthetized and a small surgical incision was made in the skin of the hindleg. 20μg/ml of WGA-HRP (Vector Laboratories; PL-1026)) was injected into individual tibialis anterior (TA, vulnerable; 3μl), gastrocnemius (GS, intermediate; 3μl), and extensor digitorum longus (EDL, resistant; 1μl) muscles. Mice were sacrificed and perfused with PBS 48 hours after surgery. Spinal cords were immediately collected, embedded in optimal cutting temperature compound (OCT) and stored at -80°C. In order to minimise contamination from other cell types when performing laser capture microdissection, frozen sections (35 μm) were mounted onto PEN-membrane slides (Applied Biosystems, Foster City, CA). Tissue sections were fixed in cold acetone and then dehydrated using a cold graded ethanol series (75%, 95% and 100% for 30 seconds), and xylene for 3 min. Dehydrated sections were developed with NovaRED Peroxidase (HRP) Substrate (Vector Laboratories, Burlingame, CA) for 3 min. Those sections identified positively for retrograde labelling were then transferred to the Arcturus Veritas laser capture microdissection system (Applied Biosystems) for isolation of labelled MN soma.

Boyd P.J., Tu W.Y., Shorrock H.K., Groen E.J., Carter R.N., Powis R.A., Thomson S.R., Thomson D., Graham L.C., Motyl A.A., Wishart T.M., Highley J.R., Morton N.M., Becker T., Becker C.G., Heath P.R, & Gillingwater T.H. (2017). Bioenergetic status modulates motor neuron vulnerability and pathogenesis in a zebrafish model of spinal muscular atrophy. PLoS Genetics, 13(4), e1006744.

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Single-Cell DNA Extraction from FFPE Samples

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Surgically resected specimens from 18 patients were acquired through collaboration with Columbia University Irving Medical Center with IRB approval under protocol number AAAS6152 and with the Johns Hopkins Department of Pathology with IRB approval under protocol number NA_00019017. Waivers of consent were granted for both protocols AAAS6152 and NA_00019017.
Frozen tissue samples were cryo-sectioned at 5 – 9 uM thick onto PEN membrane slides (Applied Biosystems cat. #LCM0522) and stored at −80°C. Slides were fixed briefly in 70% ethanol, then stained with cresyl violet acetate (32 (link)) or hematoxylin and eosin. Immediately following staining, laser-capture microdissection for the appropriate cell compartment targeted was performed using Zeiss PALM Microbeam on 10X or 20X magnification. The number of pooled cuts per cell type needed to reach a desired input of 20 – 50 ng DNA for WGBS varied for each sample. Genomic DNA was extracted using QIAamp DNA Micro Kit (Qiagen cat. #56304) according to manufacturer’s instructions for the “Isolation of Genomic DNA from Laser-Microdissected Tissues” protocol, with carrier RNA inclusion. To increase final DNA yield, samples were eluted twice, using the eluate from the first elution for the second, incubating for 5 min each before centrifugation.

Gosslar U., Jonas P., Luz A., Lifka A., Naor D., Hamann A, & Holzmann B. (1996). Predominant role of alpha 4-integrins for distinct steps of lymphoma metastasis.. Proceedings of the National Academy of Sciences of the United States of America, 93(10), 4821-4826.

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Laser Capture Microdissection of Prostate Xenografts

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Arcturus XT Laser Capture Microdissection system (Applied Biosystems, Grand Island, NY) was used to isolate the epithelial and stromal tissue compartments of the human prostate xenografts. Tissue embedded in O.C.T. medium was cut at 7 μm on the cryostat (Leica Microsystems Inc., Buffalo Grove, IL) and placed onto polyethylene naphthalate (PEN) membrane slides (Applied Biosystems). Slides were stored at −80°C until microdissection processing. Tissue sections were processed, rehydrated, and stained according to manufacturer’s instructions. Epithelial tissue was captured using the infrared system option and collection of stromal tissue immediately followed using the ultra-violet laser system. Isolated tissue was captured on Arcturus Capsure Macro LCM Caps (Applied Biosystems) and DNA was isolated from caps using the QIAamp DNA Micro Kit (Qiagen).

Saffarini C.M., McDonnell-Clark E.V., Amin A., Huse S.M, & Boekelheide K. (2015). Developmental Exposure to Estrogen Alters Differentiation and Epigenetic Programming in a Human Fetal Prostate Xenograft Model. PLoS ONE, 10(3), e0122290.

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Laser-Assisted RNA Isolation from FFPE

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Tumor and normal cells from FFPE tissue, see tissue specifications above, were Laser Capture Microdissected (LCM) using an Arcturus LCM system (ThermoFisher Scientific, Waltham, MA, USA). 10 µm sections of tissue were mounted on Pen Membrane slides (Applied Biosystems, Foster City, CA, USA). Sections were stained with cresyl violet, using the LCM staining kit (Ambion, Foster City, CA, USA/Life Technologies, Carlsbad, CA, USA) with cresyl violet according to the manufacturer’s instructions. After tissue sections had been collected and transferred to the collection cap, the cap was immediately transferred and clicked on to an Eppendorf tube containing the 100 µL of the Lysis Solution used in the first step of the RNAqueous^®-Micro Kit (ThermoFisher Scientific). The tube was inverted to ensure complete coverage of the dissected cells in the buffer. RNA isolation was performed according to the manufacturer’s instructions.

Døssing K.B., Kjær C., Vikeså J., Binderup T., Knigge U., Culler M.D., Kjær A., Federspiel B, & Friis-Hansen L. (2018). Somatostatin Analogue Treatment Primarily Induce miRNA Expression Changes and Up-Regulates Growth Inhibitory miR-7 and miR-148a in Neuroendocrine Cells. Genes, 9(7), 337.

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Tissue Sectioning and Islet Isolation

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All equipment, including microtome, blades, water bath, brushes, and polyethylene naphthalate membrane (PEN-membrane) slides (Applied Biosystems, Foster City, Calif), was sprayed with RNase Away (ThermoFisher) before tissue sectioning. Tissue sections were sliced at a 12-μm thickness (separated by 36 μm), deparaffinized, rehydrated, and stained with hematoxylin and eosin. Slides were air dried, followed by brief incubation on a hot plate and immediately taken to the Leica LMD6000 microscope (Buffalo Grove, Ill) for islet isolation as previously described.^{37 (link)}

Benner S.E., Walter D.L., Thuma J.R., Courreges M., James C.B., Schwartz F.L, & McCall K.D. (2022). Toll-Like Receptor 3 Is Critical to the Pancreatic Islet Milieu That Is Required for Coxsackievirus B4–Induced Type 1 Diabetes in Female Nonobese Diabetic Mice. Pancreas, 51(1), 48-55.

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Laser Capture Microdissection of Amyloid Plaques

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Fresh frozen TgCRND8 brains (n = 12; 6 months old) were sectioned axially (12 μm) and freeze mounted onto PEN-membrane slides (Applied Biosystems). Slides were kept on dry ice until fixation in 75% ethanol for 30 min at room temperature followed by 5 min incubation in a 1% Thioflavin-S solution (Sigma). Sections were then dehydrated in 70% ethanol for 5 min, 90% ethanol for 2 min, and 100% ethanol for 2 min. Slides were air-dried and stored at room temperature (RT) in a desiccator until LCM. Sections were cut on the laser capture system within 24 h of staining. An Arcturus laser capture system was used to gather plaque areas (defined as tissue area extending ~ 100 μm beyond the perimeter of the Thio-S positive plaque) and non-plaque areas from TgCRND8 tissue sections. For each TgCRND8 sample, 127 ± 50 plaque areas were isolated, lifted onto Arcturus caps, and processed according to manufacturer’s directions using a PicoPure Arcturus RNA extraction kit (Applied Biosystems). Non-plaque areas were gathered from the remaining Thio-S negative tissue parenchyma.

Rothman S.M., Tanis K.Q., Gandhi P., Malkov V., Marcus J., Pearson M., Stevens R., Gilliland J., Ware C., Mahadomrongkul V., O’Loughlin E., Zeballos G., Smith R., Howell B.J., Klappenbach J., Kennedy M, & Mirescu C. (2018). Human Alzheimer’s disease gene expression signatures and immune profile in APP mouse models: a discrete transcriptomic view of Aβ plaque pathology. Journal of Neuroinflammation, 15, 256.

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Cryostat Tissue Sectioning and Histochemistry

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Cryostat sections (20 μm) were mounted onto superfrost glass slides or PEN membrane slides (Thermo Fisher) and dried for 1h at room temperature. Sequential COX and succinate dehydrogenase (SDH) histochemistry was carried out to detect respiratory-deficient fibres as previously described (17 (link)). The stained sections were then used for single-cell molecular analyses.

Rygiel K.A., Tuppen H.A., Grady J.P., Vincent A., Blakely E.L., Reeve A.K., Taylor R.W., Picard M., Miller J, & Turnbull D.M. (2016). Complex mitochondrial DNA rearrangements in individual cells from patients with sporadic inclusion body myositis. Nucleic Acids Research, 44(11), 5313-5329.

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Cryosectioning and Histological Staining

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Tissue biopsies were embedded in Optimal Cooling Temperature (OCT, ThermoFisher) medium at -25°C. Sections were cut at a thickness of 20µm using a Leica Cryotome and transferred onto PEN membrane slides (ThermoFisher). For fixation, slides were treated with 70% ethanol at room-temperature for 2min. Slides were washed twice in 10% phosphate buffered saline (PBS) at room-temperature for 10s. For staining, slides were incubated in haematoxylin for 10s and rinsed twice in water. Slides were then incubated in eosin for 5s and rinsed once in water. Slides were washed twice with 70% ethanol for 5s, twice with 100% ethanol for 5s, and in xylene for 5s. Storage was at -20°C. Additional sections were stained for H&E, Masson’s Trichrome and Oil Red O by standard laboratory techniques. All slides were scanned on a Leica AT2 at ×20 magnification and a resolution of 0.5μm per pixel.

Brunner S.F., Roberts N.D., Wylie L.A., Moore L., Aitken S.J., Davies S.E., Sanders M.A., Ellis P., Alder C., Hooks Y., Abascal F., Stratton M.R., Martincorena I., Hoare M, & Campbell P.J. (2019). Somatic mutations and clonal dynamics in healthy and cirrhotic human liver. Nature, 574(7779), 538-542.

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Planarian Cryosectioning and RNA Extraction

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Planarians were killed in chilled 2% HCl in PBS (RNAse-Free) and then fixed in 100% Acetone for 1 hour at −20°C. Planarians were then incubated in 10, 20, 30% sucrose in PBS (RNAse-Free) for 20 minutes each before embedding in Tissue Freezing Medium (TFM) blocks (Ted Pella). The samples were then cryosectioned at 16 mm thickness onto PEN membrane slides (Thermo Fisher). The slides were stained with 1% cresyl violet and 1% eosin Y (Sigma) in 75% ethanol (Clément-Ziza et al., 2008 (link)). The tissue samples were dissected using Veritas Laser Capture Microdissection System (Arcturus) and captured on to Capsure HS LCM Caps (Thermo Fisher). After capture, RNA was isolated using the Arcturus PicoPure RNA isolation kit (Thermo Fisher). Ovarian tissue samples were collected from 14 worms (10 worms used for cross-sections and 4 worms used for sagittal sections) for each replicate (3 replicates total). Testis- and non-gonadal-tissue samples were collected from 6 worms for each replicate (4 worms used for cross-sections and 2 worms used for sagittal sections). RNA concentrations were determined using Qubit Fluorometer (Thermo Fisher), and RNA integrity was analyzed using 2100 Bioanalyzer Instrument (Agilent).

Khan U.W, & Newmark P.A. (2022). Somatic regulation of female germ cell regeneration and development in planarians. Cell reports, 38(11), 110525.

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LCM of AgRP Neuron-enriched ARH Samples

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LCM of AgRP neuron-enriched ARH samples was performed using fixed frozen 40-μm coronal tissue sections from male and female P12 +/+ and F/F SynTom+ mice^{61 (link)}. Sections were incubated overnight at room temperature in blocking buffer containing the rabbit anti-RFP (Abcam ab623421, 1:1000) primary antibody. Following this, sections were washed and developed using the rabbit-specific HRP/DAB detection kit (Abcam ab64261) according to the manufacturer’s directions. Sections were mounted on PEN membrane slides (ThermoFisher) prior to LCM. LCM was performed on an ArcturusXT LCM system (ThermoFisher) using the dual laser mode to microdissect and retrieve the AgRP neuron-enriched mediobasal ARH with immunohistochemical guidance. Microdissections were collected onto CapSure Macro LCM caps (Thermo Fisher) and DNA was extracted, bisulfite converted, and pyrosequenced as described above.

MacKay H., Scott C.A., Duryea J.D., Baker M.S., Laritsky E., Elson A.E., Garland Jr. T., Fiorotto M.L., Chen R., Li Y., Coarfa C., Simerly R.B, & Waterland R.A. (2019). DNA methylation in AgRP neurons regulates voluntary exercise behavior in mice. Nature Communications, 10, 5364.

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