gp120 total IgG antibodies were measured by ELISA. ELISA plates (Nunc Maxisorp 96-well plate) were coated with 100 μl of 500 ng ml−1 SIVmac251-M766 gp120 protein per well in 50 mM sodium bicarbonate buffer pH 9.6 and incubated overnight at 4 °C. Plates were blocked with 200 μl PBS Superblock (Thermo Fisher Scientific) for 1 h at room temperature. Plasma samples were serial-diluted with sample diluent (Avioq), and 100 μl of diluted plasma was added to the wells. Plates were covered and incubated for 1 h at 37 °C, washed six times with PBS Tween 20 (0.05%) and incubated with 100 μl of anti-human HRP diluted at 1:120,000 in sample diluent (Avioq) for 1 h covered at 37 °C. The plates were washed six times. Plates were developed using 100 μl K-Blue Aqueous substrate (Neogen) to all wells and incubated for 30 min at room temperature. The reaction was stopped by the addition of 100 μl of 2 N sulfuric acid to all wells and the plate was read at 450 nm on a Molecular Devices E-max plate reader.
Superblock pbs
Manufactured by Thermo Fisher Scientific
Sourced in United States
SuperBlock PBS is a solution used for blocking in immunoassays and Western blotting procedures. It is designed to reduce non-specific binding and background signal in these applications.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions)
Goat anti rabbit alexa 488, by Thermo Fisher Scientific (2 mentions)
Automated epifluorescence imaging of the array, by Zeiss (2 mentions)
Cellcelector, by Thermo Fisher Scientific (2 mentions)
Emax plate reader, by Molecular Devices (1 mentions)
K blue aqueous substrate, by Neogen (1 mentions)
Maxisorp 96 well plate, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Eclipse ti u microscope, by Nikon (1 mentions)
Trans blot transfer system, by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Amicon ultra centrifugal filter unit, by Merck Group (1 mentions)
Sample buffer, by Bio-Rad (1 mentions)
Clarity western ecl blotting substrate, by Bio-Rad (1 mentions)
Hrp conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Tgx precast gel, by Bio-Rad (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
All in one fluorescence microscope, by Keyence (1 mentions)
Collagenase type 1, by Merck Group (1 mentions)
12 protocols using superblock pbs
1
ELISA for SIVmac251-M766 gp120 Antibodies
2
Fibronectin Immunofluorescence Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were cultured on glass coverslips in 24-well plates. At the end of the experiment, cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) and kept at 4 °C for 30 minutes. After PBS washing, cells were incubated with 0.5% Triton X-100 (Fisher Scientific, Pittsburgh, PA) in PBS at room temperature for 30 minutes, and then blocked with PBS Superblock (Thermo Scientific, Rockford, IL). Cells were then immunolabelled with primary antibody: rabbit polyclonal fibronectin antibody (1:200, EMD Millipore, Billerica, MA, USA; catalog # AB1945) and incubated at 4 °C overnight. Cells incubated without primary antibody served as a negative control. Following the incubation, cells were washed three times with PBS and further incubated for 1.5 hours at room temperature with the secondary antibody (Alexa goat anti-rabbit 488; 1:500; Thermo Scientific, Rockford, IL). After PBS washing, glass coverslips with cells were then mounted on ProLong gold anti-fade reagent with DAPI (Invitrogen-Molecular Probes, Carlsbad, CA, USA). All images were taken with a Nikon Eclipse Ti-U microscope with Nuance imaging system. All antibodies used in this study were validated and characterized previously56 ,79 (link).
3
Western Blot Analysis of cNK-2 Peptide Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Collected supernatants from COS-7 cells were concentrated about 20-fold by using Amicon Ultra Centrifugal Filter Unit (MilliporeSigma). Concentrated samples were mixed with an equal volume of sample buffer (Bio-Rad), heated for 5 min at 95°C, resolved on a TGX Precast gel (Bio-Rad) and electroblotted onto polyvinyl difluoride membranes using a Trans-Blot Transfer System (Bio-Rad). The membranes were blocked with PBS SuperBlock (Thermo Fisher Scientific) Blocking Buffer for 1 hr, washed with 0.1% Tween 20 in PBS (PBS-T), and probed with anti-serum (1:100) obtained from rabbits immunized with cNK-2, cNK-lysin-derived peptide (aa101–127, RRQRSICKQLLKKLRQQLSDALQNNDD), followed by HRP-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific). The membranes were washed 5 times with PBS-T, visualized using a Clarity ECL Western Blotting Substrate (Bio-Rad), and detected using the ChemiDoc Imaging System (Bio-Rad).
4
Immunofluorescence Staining of Extracellular Matrix
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were cultured on glass coverslips in 24-well plates. At the end of the experiment, cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) and kept at 4°C for 30 minutes. After PBS washing, cells were incubated with 0.5% Triton X-100 (Fisher Scientific, Pittsburgh, PA, USA) in PBS at room temperature for 15 minutes, and then blocked with PBS Superblock (Thermo Fisher Scientific). Cells were then immunolabelled with primary antibodies: rabbit polyclonal FN antibody (1:200, catalog No. AB1945; EMD Millipore, Billerica, MA, USA) or rabbit polyclonal collagen I antibody (1:200, catalog No. NB600-408; Novus Biologicals, Centennial, CO, USA) and incubated at 4°C overnight. Cells incubated without primary antibody served as a negative control. Following the incubation, cells were washed three times with PBS and further incubated for 1.5 hours at room temperature with the secondary antibodies (1:500, Alexa goat anti-rabbit 488; Thermo Fisher Scientific). After PBS washing, glass coverslips with cells were then mounted on ProLong gold anti-fade reagent with DAPI (Invitrogen-Molecular Probes, Carlsbad, CA, USA). All images were taken with a Keyence all-in-one fluorescence microscope (Itasca, IL, USA). All antibodies used in this study have been validated and characterized previously.66 (link),71 (link)
5
Visualizing NMJ Microtubules in Muscle Fibers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The flexor digitorum brevis (FDB) muscles were harvested bilaterally from anesthetized WT and mdx mice. Single myofibers were enzymatically isolated in DMEM supplemented with 0.2 % FBS, 1 µl/ml gentamicin, and 4 mg/ml type I collagenase (Sigma, C0130) for 1 h at 37 °C as previously described [27 (link)].
Myofibers were plated on extracellular matrix (ECM; Sigma E1270)-coated imaging dishes (Matek, P35G-1.0-14-C), fixed with 4 % paraformaldehyde, permeabilized with 0.1 % Triton X-100 in PBS, blocked in Superblock PBS (Thermo Scientific), stained with BTX-594 (Molecular Probes, B13423) and then labeled with an antibody to α-tubulin conjugated to Alexa Fluor 488 (anti-mouse; Invitrogen 32-2588). Digital images were obtained using a Zeiss 510 confocal laser-scanning microscope. Laser intensity was adjusted on a sample-to-sample basis to maximize the amount of microtubules that are visualized. A 14-image Z-stack was taken at 1 µm intervals to account for total depth of the NMJ, and Image-J (NIH) was used to form a composite image. Background was subtracted uniformly, and the image was transformed into a binary image. The motor-endplate was outlined and the image cropped to isolate microtubule labeling at the NMJ. Total area of pixels at the endplate of myofibers was then quantified in Image J.
Myofibers were plated on extracellular matrix (ECM; Sigma E1270)-coated imaging dishes (Matek, P35G-1.0-14-C), fixed with 4 % paraformaldehyde, permeabilized with 0.1 % Triton X-100 in PBS, blocked in Superblock PBS (Thermo Scientific), stained with BTX-594 (Molecular Probes, B13423) and then labeled with an antibody to α-tubulin conjugated to Alexa Fluor 488 (anti-mouse; Invitrogen 32-2588). Digital images were obtained using a Zeiss 510 confocal laser-scanning microscope. Laser intensity was adjusted on a sample-to-sample basis to maximize the amount of microtubules that are visualized. A 14-image Z-stack was taken at 1 µm intervals to account for total depth of the NMJ, and Image-J (NIH) was used to form a composite image. Background was subtracted uniformly, and the image was transformed into a binary image. The motor-endplate was outlined and the image cropped to isolate microtubule labeling at the NMJ. Total area of pixels at the endplate of myofibers was then quantified in Image J.
6
Evaluating PDT and APE1 Inhibitor Effects
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The experiment was performed as described in [6 (link)]. Cells seeded in 24-well plates were cultured until 70% confluence was reached and treated with (1) PDT, (2) APE1 inhibitor, or (3) PDT with APE1 inhibitor. Untreated cells were used as a control. Afterwards, cells were washed 3 times with PBS, fixed with 3.7% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100 in PBS and again washed 3 times with PBS. Subsequently, cells were blocked in SuperBlock (PBS) (Thermo Scientific) with 0.025% Triton X-100, washed 5 times with PBS containing 0.5% BSA, and incubated overnight with primary antibodies (Table 1 ). The next day, cells were washed 5 times with PBS containing 0.5% BSA and incubated for 1 h at room temperature in the dark with secondary antibodies (Table 1 ) or phalloidin-iFluor 647 (Abcam) diluted 1:1000 in PBS containing 0.5% BSA.
7
Single-Cell Isolation and Retrieval from CTC-Enriched Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolation of CTCs from the enriched samples from the MagSweeper was performed using either the nanowell-based method with automated imaging and robotic retrieval of single cells, or a 6-well dish with manual imaging and pipetting. For the nanowell-based approach, enriched samples were loaded into the wells of a 1 × 3 inch polydimethylsiloxane nanowell device containing a 24 × 72 array of 7 × 7 wells, each of the dimensions 50 × 50 × 50 µm (Supplementary Fig. 2 )34 (link). Automated epifluorescence imaging of the array was performed (Zeiss) and images were processed using a custom software program. Following manual review of candidate cells via a custom CTC analysis software (EVA), candidate CTCs (DAPI− CD45− EpCAM+) were retrieved from individual wells of the device using an automated robotic micromanipulator (CellCelector, ALS) and deposited within 3 µL droplets of Superblock/PBS (Thermo Scientific) into empty wells of a 96 well PCR plate. For the manual approach, candidate CTCs (DAPI− CD45− EpCAM+) were recovered from Superblock/PBS by pipetting 3 µL into a 96 well PCR plate. PCR plates was frozen down at −80 °C until ready for further processing.
8
Quantitative Binding Assay for Amyloid Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plates were coated with 30 µL/well of 0.5 µM PrP23–231, PrP91–231, or PrP119–231 and incubated for 1 h at 37 °C and 300 RPM. Plates were washed three times with 100 µL/well PBS + 0.05% Tween-20 (PBST) and blocked with 100 µL/well of SuperBlock PBS (ThermoFisher, Waltham, MA) at room temperature (RT) for 2 h and 300 RPM. Plates were washed three times with 100 µL/well PBST before 30 µL/well of different proteins diluted in PBST + 0.5% BSA were added to the plates and incubated at RT for 1 h and 300 RPM. Plates were washed, and bound Aβ, αSyn, tau were detected with 30 µL/well 3D6, 2F12 and HJ8.5, respectively, diluted in PBST + 0.5% BSA at RT for 1 h and 300 RPM. Thereafter, bound antibodies were detected with 30 µL/well horseradish peroxidase (HRP)-conjugated secondary antibodies (diluted 1:15,000 in PBST + 1% BSA) at RT for 1 h and 300 RPM. After 5 × 100 µL/well washes with PBST, plates were developed.
9
Immunofluorescence Staining of Biotinylated Extracellular Vesicles
10
Single-Cell Isolation and Retrieval from CTC-Enriched Samples
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!