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Poly d lysine coverslip

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Poly-D-lysine coverslips are a type of lab equipment used to facilitate cell adhesion and growth. They provide a positively charged surface that promotes the attachment of cells to the coverslip.

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Lab products found in correlation

Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions) Lsm 510 meta confocal microscope, by Zeiss (1 mentions) Antifade mounting medium with dapi, by Vector Laboratories (1 mentions) Lsm 800, by Zeiss (1 mentions) Halocarbon oil 700, by Merck Group (1 mentions) Quantigene viewrna ish cell assay kit, by Thermo Fisher Scientific (1 mentions) Antifade mounting medium, by Vector Laboratories (1 mentions)

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Poly-D-lysine (62 citations) Coverslip (5 citations)

6 protocols using poly d lysine coverslip

1

Retinal Preparation for Electrophysiology

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Mice were dark-adapted overnight and anesthetized by intraperitoneal injection of 2,2,2-Tribromoethanol. Then, under dim red light, a 30 gauge needle was used to mark the nasal corneal margin. Mice were killed by cervical dislocation and the eyes were enucleated and transferred a petri dish with carbogenated (95% O2/5% CO2) Ames’ medium. A large relieving cut was made in the nasal margin of the eye cup prior to removing the retina from the eye cup to keep track of retinal orientation. Retinas were then mounted onto a 12 mm poly-D-lysine coverslip (Corning). The coverslip was then mounted directly onto a recording chamber, anchored using a platinum ring with nylon mesh and placed on an electrophysiology rig. The retina was superfused with carbogenated Ames’ medium (30–32°C) at 5–7 mL/min for at least 30 minutes prior to recording. For experiments in which retinal location was not documented, retinas were sliced in half and incubated in a beaker with carbogenated Ames’ medium (26°C) prior to mounting them on the electrophysiology rig.
Sonoda T., Okabe Y, & Schmidt T.M. (2019). Overlapping morphological and functional properties between M4 and M5 intrinsically photosensitive retinal ganglion cells. The Journal of comparative neurology, 528(6), 1028-1040.
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2

All-in-one dLwaCas13a-msfGFP Plasmid

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pC035-dLwaCas13a-msfGFP plasmid was purchased from Addgene (Addgene #91925) and used directly for no-crRNA control experiments. We also modified dLwaCas13a-msfGFP into an all-in-one plasmid that also included an crRNA-expressing cassette cloning from LwaCas13a guide expression backbone with U6 promoter, with crRNAs targeted to endogenous gene via Gibson assembly to generate dLwaCas13a-msfGFP-crRNA plasmid. To make the AcrVIA plasmids, we amplified an AcrVIA5 cassette and incorporated in pEJS481-pCDest2-AcrE2-mTagBFP2-IRES (Addgene #85746) to replace the AcrE2 by Gibson assembly. HEK293T cells were plated in 24-well tissue culture plates on poly-d-lysine coverslip (Corning) and transfected by LipofectAmine 3000 (Thermo Fisher Scientific). After 48 h incubation, cells were washed with PBS, and fixed in 4% paraformaldehyde for 15 min. Images were observed under a Zeiss LSM 510 Meta Confocal Microscope. Only cells that exhibited mTagBFP2 and msfGFP fluorescence were assessed for the presence or absence of co-localizing dLwaCas13a-msfGFP foci, and such imaged cells were included in the quantifications.
Lin P., Qin S., Pu Q., Wang Z., Wu Q., Gao P., Schettler J., Guo K., Li R., Li G., Huang C., Wei Y., Gao G.F., Jiang J, & Wu M. (2020). CRISPR-Cas13 inhibitors block RNA-editing in bacteria and mammalian cells. Molecular cell, 78(5), 850-861.e5.
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3

Visualizing ACTB mRNA Translocation

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HEK293FT cells were plated in 24-well tissue culture plates on poly-D-lysine coverslips (Corning) and transfected with 150ng dCas13a-NF vector and 300 ng guides for imaging ACTB. For translocation experiments, cells were fixed with 4% PFA and permeabilized with 0.2% Triton X-100 after 48 hours and mounted using anti-fade mounting medium with DAPI (Vectashield). Confocal microscopy was performed using a Nikon Eclipse Ti1 with Andor Yokagawa Spinning disk Revolution WD system.
Nuclear export of dCas13a-NF-msfGFP with guides targeting ACTB mRNA was analyzed by measuring the average cytoplasmic and nuclear msfGFP fluorescence and comparing the ratio across many cells between targeting and non-targeting conditions.
Abudayyeh O.O., Gootenberg J.S., Essletzbichler P., Han S., Joung J., Belanto J.J., Verdine V., Cox D.B., Kellner M.J., Regev A., Lander E.S., Voytas D.F., Ting A.Y, & Zhang F. (2017). RNA targeting with CRISPR-Cas13a. Nature, 550(7675), 280-284.
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4

Dual-Color Calcium Imaging in Fictive Preps

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For dual-color and single-color calcium imaging in fictive preps, freshly dissected brains were mounted on 12 mm round Poly-D-Lysine Coverslips (Corning BioCoat) in HL3.1 saline (de Castro et al., 2014 (link)), which were then were placed on 25 mm ×75 mm glass slides to be imaged with a 40 × objective on an upright Zeiss LSM-800 confocal microscopy. To simultaneously image two different neurons expressing GCaMP6m we imaged neuron-specific regions of interest (ROI). In addition, we imaged two neurons differentially expressing GCaMP6m and jRCaMP1b. Image data were imported into Fiji (https://imagej.net/fiji) and GCaMP6m and jRCaMP1b channels were separated. The ΔF/F0 of each ROI was calculated as (F-F0)/F0, where F0 was averaged over ~1 s immediately before the start of the forward or backward waves in each ROI.
Zarin A.A., Mark B., Cardona A., Litwin-Kumar A, & Doe C.Q. (2019). A multilayer circuit architecture for the generation of distinct locomotor behaviors in Drosophila. eLife, 8, e51781.
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5

Dual-Color Calcium Imaging in Fictive and Intact Preps

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For dual-color and single-color calcium imaging in fictive preps, freshly dissected brains were mounted on 12 mm round Poly-D-Lysine Coverslips (Corning BioCoat) in HL3.1 saline, which were then were placed on 25 mm ×75 mm glass slides to be imaged with a 40 × objective on an upright Zeiss LSM-800 confocal microscopy. To do calcium imaging in intact animals (e.g. Figure 5D’), a second or third instar larva was washed with distilled water, then moved into a drop of halocarbon oil 700 (Sigma, St. Louis, MO) on the slide. A 22 mm × 40 mm cover glass was put on the larva and pressed gently to restrict larval locomotion. The larva was mounted ventral side up so that the ventral nerve cord could be imaged using 40 × objective on an upright Zeiss LSM800 confocal microscope. To simultaneously image two different neurons expressing GCaMP, we imaged neuron-specific regions of interest (ROI). In addition, we imaged two neurons using neuron-specific GCaMP6m and jRCaMP1b. Image data were imported into FijI (https://imagej.net/fiji) and GCaMP6m and jRCaMP1b channels were separated. The ΔF/F0 of each ROI was calculated as (F-F0)/F0, where F0 was averaged over ~1 s immediately before the start of the forward or backward waves in each ROI.
Carreira-Rosario A., Zarin A.A., Clark M.Q., Manning L., Fetter R.D., Cardona A, & Doe C.Q. (2018). MDN brain descending neurons coordinately activate backward and inhibit forward locomotion. eLife, 7, e38554.
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6

CRISPR-Cas13a RNA Imaging in HEK293FT

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HEK293FT cells were plated in 24-well tissue culture plates on poly-D-lysine coverslips (Corning) and transfected with 75 ng dCas13a-NF vector and 250 ng guides for imaging ACTB. After 48 hours, cells were fixed with 4% PFA for 45 minutes. The QuantiGene view RNA ISH Cell assay kit (Affymetrix) was used for performing the FISH on the cell samples and the protocol was followed as described by the manufacturer. After finishing the FISH procedure, coverslips were mounted using anti-fade mounting medium (Vectashield). Confocal microscopy was performed using a Nikon Eclipse Ti1 with Andor Yokagawa Spinning disk Revolution WD system.
Abudayyeh O.O., Gootenberg J.S., Essletzbichler P., Han S., Joung J., Belanto J.J., Verdine V., Cox D.B., Kellner M.J., Regev A., Lander E.S., Voytas D.F., Ting A.Y, & Zhang F. (2017). RNA targeting with CRISPR-Cas13a. Nature, 550(7675), 280-284.
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