Nitrocellulose filter paper

Manufactured by Merck Group

Sourced in Germany, United States

Nitrocellulose filter paper is a laboratory filtration product used for the separation and isolation of various biomolecules, such as proteins, nucleic acids, and cells. It is a thin, porous membrane made from nitrocellulose that provides efficient filtration and retention of analytes. The product is designed for use in a range of laboratory applications, including Western blotting, dot blotting, and ELISA techniques.

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Lab products found in correlation

Helios gene gun, by Bio-Rad (3 mentions) Biopore membrane, by Merck Group (2 mentions) 1.6 μm gold particles, by Bio-Rad (2 mentions) Teflon tubing, by Bio-Rad (2 mentions) Liquid scintillation counter, by PerkinElmer (2 mentions) Ames media, by Merck Group (2 mentions) Cal 520 am, by AAT Bioquest (1 mentions) Tc 344b, by Warner Instruments (1 mentions) Mea 60, by MultiSciences Biotech (1 mentions) Gs gene linker, by Bio-Rad (1 mentions) Sp5 confocal microscope, by Leica (1 mentions) Gold particles, by Bio-Rad (1 mentions) Mouse igg1, by BD (1 mentions) Vectashield, by Vector Laboratories (1 mentions) Mouse igg2b, by Santa Cruz Biotechnology (1 mentions)

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21 protocols using nitrocellulose filter paper

Harvest and Imaging of Fetal Retinas

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On the day of the experiment, pregnant dams were deeply anesthetized via isoflurane inhalation and fetuses were harvested via a cesarean section. tdTomato-positive fetuses were identified using miner goggles (Biological Laboratory Equipment Services and Maintenance Ltd., model: GFsP-5). Fetuses were kept alive in 50 ml Falcon tubes filled with oxygenated (95% O₂ 5% CO₂) ACSF (in mM, 119 NaCl, 2.5 KCl, 1.3 MgCl₂, 1 K₂HPO₄, 26.2 NaHCO₃, 11 D-glucose, and 2.5 CaCl₂). Fetuses were then euthanized sequentially by decapitation. Eyes were immediately enucleated and retinas were dissected at room temperature in oxygenated ACSF under a dissecting microscope. Isolated retinas were mounted whole over a 1–2 mm² hole in nitrocellulose filter paper (Millipore) with the photoreceptor layer side down and transferred to the recording chamber of a two-photon microscope for imaging. The whole-mount retinas were continuously perfused (3 ml/min) with oxygenated ACSF warmed to 32–34°C by a regulated inline heater (TC-344B, Warner Instruments) for the duration of the experiment. Additional retina pieces were kept in the dark at room temperature in ACSF bubbled with 95% O2, 5%CO2 until use (maximum 8 hr).
For the calcium imaging experiments, retinas were bath loaded with the calcium indicator Cal 520 AM (AAT Bioquest) for 1–2 hr at 32°C.

Voufo C., Chen A.Q., Smith B.E., Yan R., Feller M.B, & Tiriac A. (2023). Circuit mechanisms underlying embryonic retinal waves. eLife, 12, e81983.

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Retinal Flat-Mount and Slicing Protocols

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All procedures were approved by the UC Berkeley Animal Care and Use Committee. After adult fish were euthanized in MS-222, the dark-adapted retinas were isolated from the eyecups with the RPE removed, and kept in darkness in bubbled bicarbonate buffered saline. For flat-mount preparation, retinas were mounted onto a Biopore^™ membrane (MILLIPORE) with photoreceptors facing the membrane^{46 (link)}. For slice preparation, retinas were mounted onto nitrocellulose filter paper (pore size 0.8 μm, MILLIPORE) with retinal ganglion cells facing the paper. Retinas were sliced to 200 μm in thickness using a tissue slicer^{27 (link)}. Slices were counter-stained with 2.5 μM sulforhodamine 101 for Fig. 1c. For Supplementary Fig. 1, hippocampal neurons were prepared and transfected using standard protocols.

Wang T.M., Holzhausen L.C, & Kramer R.H. (2014). Imaging an optogenetic pH sensor reveals that protons mediate lateral inhibition in the retina. Nature neuroscience, 17(2), 262-268.

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Immunolabeling of Whole-Mount Retina

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Mice were euthanized by isoflurane overdose or carbon dioxide followed by cervical dislocation. The mice were enucleated and the retinas were dissected and mounted on nitrocellulose filter paper (Millipore) in bicarbonate-based Ames solution equilibrated with 95% O₂/5% CO₂. The orientation and sidedness of retinas were noted so that dorsal-ventral and temporal-nasal axes could be distinguished in the whole mount retina. The retinas were fixed in 4% paraformaldehyde for 15 min or 2% paraformaldehyde for 20 min at room temperature, rinsed in PBS, pH 7.42, then immersed in blocking solution (5% normal donkey serum, 0.5% Triton X-100 in PBS) overnight, incubated in primary antibodies for 5 days at 4°C, then rinsed in PBS and incubated in secondary antibodies for 1 day at 4°C, and rinsed with PBS and mounted with Vectashield underneath a coverslip. For quantification of cell types in sections, the whole eye was fixed in 4% paraformaldehyde for 90 min, 20 μm frozen sections were rinsed in PBS, incubated in primary antibodies for day at 4°C, rinsed with PBS, incubated in secondary antibody for 1 hr at room temperature, rinsed with PBS, and mounted with Vectashield underneath a coverslip. Reagents are listed in the Key Resources Table.

Care R.A., Kastner D.B., De la Huerta I., Pan S., Khoche A., Santina L.D., Gamlin C., Tomas C.S., Ngo J., Chen A., Kuo Y.M., Ou Y, & Dunn F.A. (2019). Partial Cone Loss Triggers Synapse-Specific Remodeling and Spatial Receptive Field Rearrangements in a Mature Retinal Circuit. Cell reports, 27(7), 2171-2183.e5.

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Retinal Flat-Mount and Slicing Protocols

Cited 2 times

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Wang T.M., Holzhausen L.C, & Kramer R.H. (2014). Imaging an optogenetic pH sensor reveals that protons mediate lateral inhibition in the retina. Nature neuroscience, 17(2), 262-268.

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Imaging RGCs Dynamics in Retinal Whole-Mounts

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The time-lapse images of RGCs were taken from whole-mount retina preparations as previously described (Xu et al., 2010 (link)). Retinas of Thy1-YFP mice, BD:YFP mice and JamB:YFP mice were used for ex vivo imaging of αRGCs, BD-RGCs and J-RGCs, respectively. Retinas were isolated from Thy1-YFP, BD:YFP and JamB:YFP mice in oxygenated extracellular solution that contained (in mmol/L) NaCl 124, KCl 2.5, CaCl2 2, MgCl2 2, NaH2PO4, 1.25, NaHCO3 26, and glucose 22 (pH 7.35 with 95% O2 and 5% CO2), mounted on nitrocellulose filter paper (Millipore Corp), placed in a recording chamber and continuously perfused at 32°C. Image stacks were taken using a two-photon image system (Prairie Technologies, Inc., Middleton, WI, United States) immediately before bath application of NMDA. After 10 min of bath application of 200 nmol/L NMDA, the retinas were continuously perfused with the oxygenated extracellular solution and the cells were imaged every 1 h for 7 h. The dendritic density of imaged cells is measured using a Sholl analysis (Xu et al., 2010 (link)) and the dendritic density after NMDA application was normalized to pre-NMDA application (0 h).

Christensen I., Lu B., Yang N., Huang K., Wang P, & Tian N. (2019). The Susceptibility of Retinal Ganglion Cells to Glutamatergic Excitotoxicity Is Type-Specific. Frontiers in Neuroscience, 13, 219.

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Retinal Ganglion Cell Electrophysiology in Mice

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Nineteen male C57Bl/6J mice were kept on a regular light/dark cycle and experiments were performed diurnally at 3–4 months of age. Prior to euthanization, mice were dark adapted for at least 90 min. Eyes were removed under infrared illumination using night vision (Nitemare, BE Meyers, Oregon) and their retinas were dissected in a dish containing carboxygenated recording solution. Retinas were placed ganglion cell side up onto nitrocellulose filter paper (0.45 μm HA, Millipore) and transferred onto an electrode array where the preparation was retained with a plastic ring.
The retina was kept at 35.6°C and perfused at 2 mL/min with prewarmed and carboxygenated (95% O₂, 5% CO₂) recording medium (in mmol/L: NaCl, 124; KCl, 2.5; CaCl₂, 2; MgCl₂, 2; NaH₂PO₄, 1.25; NaHCO₃, 26; and glucose, 22) at pH 7.35 (Tian and Copenhagen 2003). The multielectrode array (MEA‐60, Multichannel Systems, Tübingen Germany) had 60 electrodes spaced 100 μm apart and with diameters of 10 μm. Ganglion cell action potentials were recorded at 20 Khz and prefiltered with a 0.1 Hz high‐pass hardware filter.

Cowan C.S., Sabharwal J, & Wu S.M. (2016). Space–time codependence of retinal ganglion cells can be explained by novel and separable components of their receptive fields. Physiological Reports, 4(17), e12952.

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Biolistic Transfection of Retinal Neurons

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Mice were anesthetized by isoflurane and euthanized by cervical dislocation. Eyes were removed and placed in oxygenated mouse ACSF (pH 7.4), containing the following (in mm): 119 NaCl, 2.5 KCl, 1.3 MgCl₂*6H₂O, 2.5 CaCl₂*2H2O, 1 NaHPO₄, 11 glucose, 20 HEPES. Retinas were isolated from the eyecup under a dissection microscope and mounted onto nitrocellulose filter paper (Millipore). DNA-coated gold particles were prepared by coating 12.5 mg of 1.6 μm gold particles (Bio-Rad) with 20 μg of CMV:CFP and 7 μg of CMV:PSD95-YFP plasmids. A Helios gene gun (Bio-Rad) was used to biolistically deliver plasmid-coated gold particles to whole-mounted retinas. A suspension of DNA-coated gold particles in ethanol was precipitated onto the inner surface of Teflon tubing (Bio-Rad) and subsequently cut into short segments (12 mm long). Gold particles were propelled onto the tissue using helium gas at 40 psi. Retinas were then transferred to an oxygenated and humidified chamber and maintained for 26 h at 32°C, allowing fluorescent protein to be expressed sufficiently for subsequent imaging.

Della Santina L., Yu A.K., Harris S.C., Soliño M., Ruiz T.G., Most J., Kuo Y.M., Dunn F.A, & Ou Y. (2021). Disassembly and rewiring of a mature converging excitatory circuit following injury. Cell reports, 36(5), 109463.

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Retinal Dissection and Electrophysiology

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Animals were deeply anesthetized with isoflurane and decapitated. After enucleation of the eyes, retinas were dissected in oxygenated (95% O₂/5% CO₂) ACSF (119 mM NaCl, 2.5 mM KCl, 1.3 mM MgCl₂, 1 mM K₂HPO₄, 26.2 mM NaHCO₃, 11 mM D-glucose, and 2.5 mM CaCl₂) at room temperature under infrared illumination. Isolated retinas were mounted RGC side up on nitrocellulose filter paper (Millipore), and transferred to a recording chamber of an upright microscope for imaging and simultaneous electrophysiological recording. The whole-mount retinas were continuously perfused (3 ml/min) with oxygenated ACSF at 32–34°C for the duration of the experiment.

Webster C.M., Tworig J., Caval-Holme F., Morgans C.W, & Feller M.B. (2020). The Impact of Steroid Activation of TRPM3 on Spontaneous Activity in the Developing Retina. eNeuro, 7(2), ENEURO.0175-19.2020.

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Binding Kinetics of Lysozyme and bDV RNA

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Filter binding studies were performed as described in refs 4 (link) and 52 (link). The ³²P-labelled bDV RNA was obtained by in vitro transcription (stated above) in presence of a ³²P-UTP followed by DNase I digestion and precipitation using ethanol. Lysozyme was incubated with equimolar ³²P-bDV RNA in the presence of 20 mM DTT for various time intervals and filtered through pre-soaked nitrocellulose filter paper (Millipore) with pore size of 0.22 μm. The filters were dried and ³²P counts were taken in a liquid scintillation counter (Perkin Elmer). The RNA bound to the protein was retained on the filter while the free RNA passed through it. The percentage of radioactivity retained on the filter paper, calculated considering the radioactive count incorporated in the total RNA to be 100%, was plotted against time.

Pathak B.K., Mondal S., Banerjee S., Ghosh A.N, & Barat C. (2017). Sequestration of Ribosome during Protein Aggregate Formation: Contribution of ribosomal RNA. Scientific Reports, 7, 42017.

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In vitro Protein-RNA Binding Assay

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Filter binding studies were performed as described in earlier studies [26] (link). The wild-type and mutant RNA were labeled with radio-isotope [α-³²P] UTP during in vitro transcription. Reduced-denatured lysozyme or BCAII-m was incubated with equimolar radiolabeled RNA at 4°C for different time intervals. After incubation the samples were cross-linked by UV irradiation at 254 nm for 90 seconds (GS GeneLinker, Bio-Rad) and filtered through pre-soaked nitrocellulose filter paper (Millipore) with pore size of 0.22 µM. The filter papers were dried and ³²P counts were taken in a liquid scintillation counter (Perkin Elmer). The RNA bound to denatured protein was retained on the filter while the free RNA passed through it. The percentage of radioactivity retained on the filter paper was calculated and plotted against incubated time. Comparing the radioactive count incorporated in the total RNA to that on the filter, the percentage of radioactivity retained was calculated.

Pathak B.K., Mondal S., Ghosh A.N, & Barat C. (2014). The Ribosome Can Prevent Aggregation of Partially Folded Protein Intermediates: Studies Using the Escherichia coli Ribosome. PLoS ONE, 9(5), e96425.

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