Retinal punches from the macula and peripheral regions of donor eyes were fixed in 4% paraformaldehyde (PFA) in PBS for 2 hr and then transferred to PBS for 1 hr. Retinal pieces were embedded in low melting point agarose (42°C, 3% agarose (Lonza) in PBS). Tissue blocks were cooled down at 4°C until the agarose solidified. agarose blocks were trimmed and glued to the metal chuck of a vibratome (Leica, VT1200S). Vibratome sections (100 μm thick) were obtained from retinal tissues and stored in PBS at 4°C.
Agarose
Manufactured by Lonza
Sourced in United States, Switzerland
Agarose is a polysaccharide extracted from certain red seaweeds. It is commonly used as a gel matrix in various laboratory applications, such as electrophoresis and chromatography. Agarose provides a porous, inert, and hydrophilic medium for the separation and purification of biomolecules like nucleic acids and proteins.
Automatically generated - may contain errors
Lab products found in correlation
Crystal violet, by Merck Group (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Neutral red, by Merck Group (4 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (4 mentions)
Glycerol, by Merck Group (4 mentions)
Agarose, by Merck Group (3 mentions)
Ethidium bromide, by Merck Group (3 mentions)
Seaplaque agarose, by Lonza (3 mentions)
Uranyl formate, by Electron Microscopy Sciences (3 mentions)
Pcr tubes, by Avantor (3 mentions)
Carbon formvar grids, by Electron Microscopy Sciences (3 mentions)
Amicon ultra filtration devices, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Tween 20, by Merck Group (3 mentions)
Tris base, by Merck Group (3 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (3 mentions)
6 well plate, by Corning (2 mentions)
Milli q purification system, by Merck Group (2 mentions)
Mueller hinton broth (mhb), by Boster Bio (2 mentions)
Trizma base, by Merck Group (2 mentions)
75 protocols using agarose
1
Retinal tissue sectioning and preservation
2
Soft Agar Colony Formation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RPMI culture medium containing 0.3% agarose (Lonza, Basel, Switzerland) with LM05-shGFP or LM05-shNIK cells (4.0 × 104 cells/well) over a bottom layer of 0.6% agarose in RPMI culture medium were plated in each well of a 6-well plate and cultured for 3 weeks. Colonies were fixed with 4% paraformaldehyde-PBS (Fujifilm Wako Pure Chemical Corporation) for 1 h and stained with 0.005% crystal violet solution (Fujifilm Wako Pure Chemical Corporation) for 30 min. After removing the overdyed region with Milli-Q water, the colony images were acquired with a digital camera (Nikon Corporation, Tokyo, Japan), and colony numbers were calculated with ImageJ software (National Institutes of Health, MD, USA).
3
SARS-CoV-2 Neutralizing Antibody Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate the neutralizing activity of monoclonal antibodies, plaque reduction neutralizing tests for SARS-CoV-2 were performed as described previously43 (link). Briefly, twofold serially diluted mAbs ranging from 103 to 1 ng/ml and an equal volume of virus (40 pfu/well) were incubated at 37 °C for 2 h. The antibody–virus mixture was inoculated into a 24-well plate seeded with VeroE6 cells (1 × 105 cells/well) and incubated at 37 °C for 1 h, followed by an overlay of 1 ml of 0.5% agarose (Lonza). After 2 to 3 days of incubation, the cells were fixed with 4% paraformaldehyde and visualized plaques with crystal violet. Two independent experiments were performed in duplicate for each mAb. The data were fitted to a dose–response inhibition model, and the half-maximal inhibitory concentration (IC50) of each mAb was calculated using GraphPad Prism6 software.
4
Colony Formation Assay in 3D Agarose
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
One milliliter of 0.5% agarose (Lonza Japan) in RD medium was solidified in each well of a 6-well plate (Corning, NY, USA) as the bottom agarose layer and incubated for 20 min at 24°C. The 0.3% top agarose containing cells was poured over the 0.5% agarose gel (2,500 cells/well) and set at 10 min at 4°C. The plate was cultured in a humidified incubator at 37°C with 5% CO2 for 14 days, rinsed twice with PBS, and fixed with methanol for 15 min. Colonies were stained for 20 min with Giemsa’s solution (Merck, Darmstadt, Germany) diluted 1: 20 in phosphate buffer (4.7 mM KH2PO4, 2 mM Na2HPO4). Digital images of the colonies were taken using a Biozero microscope (Keyence). Colonies were counted using Image J software (NIH, USA).
5
Agarose Gel Electrophoresis Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Analysis of samples by gel imaging was performed by using a 1% agarose (Lonza) gel in TBEMg 1× buffer [40 mM tris base, 20 mM boric acid, 2 mM EDTA and 12.5 mM Mg acetate (pH 8.0)], at 80 V for 2 hours at 4°C. The gel was scanned with a Typhoon FLA9000 (GE Healthcare Life Sciences) at different wavelengths and lastly stained with ethidium bromide.
6
Genetic Diversity Analysis of Tinospora Accessions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nineteen SCoT markers were used for the genetic diversity analysis of five additional T. cordifolia accession (IC-281970, IC-471321, IC-281968, KCOP/41, KCOP/18), one accession of T. rumphii (KCB/38), and one accession of T. sinensis (KC/NS/GD-42/14). The PCR amplification conditions were the same as Paliwal et al., 2013 [23 ]. All amplified products were mixed with gel-loading dye (bromophenol blue, xylene cyanol, and sucrose) and then analyzed on 1% agarose (Lonza, USA) gel for 4 h at a constant supply of 80 V. Gel pictures were recorded using a Gel Documentation System (α Imager®, USA).
7
Immunoglobulin Gene Sequence Determination
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA was isolated from hybridoma clones using a Gentra Puregene cell kit (Qiagen). Immunoglobulin heavy and light chain genes were amplified by PCR using a Platinum Taq DNA Polymerase High Fidelity kit (Thermo Fisher Scientific) using previously described primers [27 (link),28 (link)]. Amplified DNA was run on a 1% agarose (Lonza) in TAE buffer (Corning) gel and DNA was purified from the gel using a Zymoclean gel DNA recovery kit (Zymo Research). DNA fragments were then sequenced by Sanger sequencing. For hybridomas whose productive and non-productive rearrangements were not easily separated by gel electrophoresis, the DNA was subcloned using a TOPO TA Cloning kit with the pCRII-TOPO vector (Invitrogen).
8
Soft Agar Colony Formation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DLD-1 cells were seeded at a concentration of 0.5 × 105 cells/ml in 6-well plates. Twenty-four hours later, the cells were transfected with control RNA, siR-PTBP1, siR-hnRNPA1, or siR-SRSF3 (5 nM) for 48 h. Next, 2 × 102 cells from each transfection group were suspended in 0.4% agarose (Lonza, ME, USA) in RPMI 1680 medium (Wako), supplemented with 8% (v/v) heat-inactivated FBS (Sigma-Aldrich) in 6-cm dishes (CORNING, Corning, NY, USA) as the upper layer, with 0.5% agarose in RPMI 1680 medium, supplemented with 8% (v/v) heat-inactivated FBS as the bottom layer. The cells were cultured under an atmosphere of 95% air and 5% CO2 at 37 °C for 2 weeks. The colonies were fixed with methanol (Sigma Aldrich) for 10 min and stained with 5% Giemsa’s stain solution (MUTO PURE CHEMICALS CO., LTD., Tokyo, Japan) for 30 min. After wash and dry, the colonies were counted.
9
Colony Formation Assay for MDA-MB-231 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MDA-MB-231 parental cells and BSVTK-labeled MDA-MB-231 cells were plated at low density (1000 cells per well in six-well plates) with three technical replicates per experiment. After 1 week of incubation, the colonies were stained with crystal violet overnight at 4°C. After wash steps, the plates were scanned and colonies manually counted using FiJi software. For soft agar colony formation, 3 ml of bottom agarose layer, containing 0.75% agarose (#50101, Lonza) in RPMI medium (#23400-021, Thermo Fisher Scientific) was supplemented with FBS and penicillin-streptomycin. Once the bottom layer was solidified, 25,000 MDA-MB-231 parental or LeGO cells were mixed in top agarose layer (0.45% agarose) and seeded on top of the bottom layer. The wells were covered with 3 ml of culture media and incubated 2 weeks at 37°C in a humidified atmosphere with 5% CO2. After 2 weeks, 140 μl of MTT (5 mg/ml) in PBS was added into each well, and the plates were incubated for 4 hours. Last, the plates were scanned and manually counted using FiJi software.
10
Zika Virus Plaque Assay Protocol
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!