The largest database of trusted experimental protocols

C8035

Manufactured by Merck Group
Sourced in Denmark, Japan, United States

The C8035 is a laboratory equipment product from Merck Group. It is designed for performing analytical tasks in a research or testing environment. The core function of the C8035 is to facilitate precise measurements and analyses required for scientific investigations. Further details on the intended use or specific applications of this product are not available.

Automatically generated - may contain errors

16 protocols using c8035

1

Flow Cytometry Cell Surface Binding

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cells were detached from tissue culture flasks using CellStripper dissociation reagent (Corning), quenched with PBS with calcium and magnesium (PBS+/+) supplemented to contain 2% FBS, and centrifuged at 1,500 × g at 4°C for 5 min. Cells (5 × 105 cells per sample) were stained with human anti-HS (1:750; Amsbio 370255-S), human anti-CS (1:750; Sigma C8035), human anti-Mxra8 (1 μg/ml; MBL International W040-3), or mouse anti-Mxra8 (1 μg/ml; 4E7.D10 [31 (link)]) antibodies at 4°C for 1 h. Cells were incubated with Alexa Fluor 647 antibody (1:1,000; Thermo Fischer Scientific) at 4°C for 1 h. Samples were washed twice with VDB between incubations. Samples were fixed with 1% PFA at 4°C for 5 min and analyzed by flow cytometry (LSRII flow cytometer; BD Biosciences). Binding events were gated using secondary-antibody-only control samples as the no-binding controls, and median fluorescent intensity (MFI) was determined using FlowJo V10 software.
+ Open protocol
+ Expand
2

Immunohistochemical Staining Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols
Immunohistochemical studies were performed as previously described.23 Briefly, serial 10‐μm‐thick sections were fixed in ice‐cold acetone and incubated with normal horse serum or normal goat serum for 60 min, followed by incubation with primary antibodies. After washing with phosphate‐buffered saline, sections were incubated with biotinylated horse antimouse immunoglobulin G or biotinylated horse antimouse immunoglobulin M (Vector Laboratories) for 60 min. Next, sections were washed extensively and exposed to avidin–biotin complex (Vector Laboratories) for 60 min and were then covered with diaminobenzidine for 10 min. Primary antibodies were mouse anti–HLA‐ABC (555551, Becton‐Dickinson, 1:4000), mouse anti‐CD8 (M7103, Dako, 1:150), mouse anti‐p62 (ab56416, Abcam, 1:300), mouse anti‐chondroitin sulfate proteoglycan (C8035, Sigma‐Aldrich, 1:2000), mouse anti‐keratan sulfate (PRPG‐BG‐M01, Cosmo Bio Co, 1:30), and mouse anti‐heparan sulfate (H1890, US Biological, 1:50).
+ Open protocol
+ Expand
3

Quantifying Tumor-Derived CSPG Secretion

Check if the same lab product or an alternative is used in the 5 most similar protocols
CSPGs released by the tumor cells in media were measured using an ELISA based assay67 (link). Media samples from the 3D tumor constructs were incubated overnight at 4 °C in a 96-well immuno plate (Thermo Fischer Scientific). Alongside the sample media incubation, chicken extracellular CSPGs (Millipore, CC117) were used over a range of serial dilutions for the generation of standard curves. Following washes with PBS-Tween, monoclonal anti-chondroitin sulfate antibody produced in mouse/clone CS-56, ascites fluid (Sigma–Aldrich, C8035) was added for overnight incubation at 4 °C. After the next round of washes, HRP conjugated goat anti-mouse secondary antibody (Abcam) was incubated at room temperature for 2 h. TMB (3,3′,5,5′-tetramethylbenzidine) 1-C Substrate (Fischer Scientific) was introduced following the last round of washes with PBS-Tween. Finally, after the color developed for 10 min at room temperature, the reaction was stopped with 1 N HCl. The absorbance readings were measured at 450 nm wavelength and the fresh media readings were subtracted from the sample readings. The standard curve was utilized for calculating the quantities of CSPGs released in the different conditions and reported in pg mL−1.
+ Open protocol
+ Expand
4

Western Blotting Analysis of Protein Targets

Check if the same lab product or an alternative is used in the 5 most similar protocols
Tissue samples or cerebellar slices or purified OPC cells were homogenized with RIPA lysis buffer and protein concentration was determined by Pierce BCA protein assay kit according to the manufacture’s instructions (Thermo Fisher). Then, equal amounts of protein were loaded onto 15% SDS-PAGE gels, and electrophoretically transferred to PVDF membranes (Millipore). The membranes were blocked in 0.1% TPBS buffer with 5% non-fat milk for 1 h at room temperature and probed with indicated primary antibodies overnight at 4 °C and followed by secondary antibodies conjugated to horseradish peroxidase (HRP). The following primary antibodies were used: MBP (SMI-99P, Covance, 1:1000), CS56 (C8035, Sigma, 1:1000), Laminin (L9393, Sigma, 1:1000), GAPDH (AF5718, R and D Systems, 1:1000), MMP-2 (AF1488, R and D Systems, 1:1000), MMP-10 (MAB910, R and D Systems, 1:1000), and β-actin (sc-47778, Santa Cruz, 1:1000). Enhanced chemiluminescence was performed with a West Pico Kit (Thermo Fisher) and detected by FluorChem E system (ProteinSimple, USA). The density of bands was quantified using ImageJ software (NIH). Image has been cropped for presentation (full-size image is shown in Supplementary Figs. 12, 13).
+ Open protocol
+ Expand
5

Immunohistochemical Staining of Perineuronal Nets and Parvalbumin Neurons

Check if the same lab product or an alternative is used in the 5 most similar protocols
One set of brain sections from one hemisphere of each bird was processed for both PNN and PV expression. Free-floating sections were washed 3X for 5 min in 1X TBS and then blocked for 30 min in TBS + 5% donkey serum + 0.1% Triton-X. Then the tissue was incubated overnight at 4°C in a mouse monoclonal anti-chondroitin sulfate (C8035; Sigma-Aldrich; 1:500) and a rabbit polyclonal anti-PV (ab11427; Abcam; 1:2000). Thereafter, sections were washed 3X for 5 min in TBS followed by a 2-h incubation at room temperature with secondary antibodies [donkey anti-mouse Alexa Fluor 488: 10 μl/ml (ThermoFisher); donkey anti-rabbit Alexa Fluor 594: 5 μl/ml (ThermoFisher)] in TBS + 0.1% Triton-X. The tissue was then washed 3X for 5 min in TBS and transferred to TBS before mounting. Sections were coverslipped with Prolong Gold Antifade (Life Technologies, 2491361).
Mouse brains were processed in a similar manner as finch brains, albeit with different primary antibodies (Cisneros-Franco et al., 2018 (link)). For mouse sections, PV neurons were stained using same PV antibody used in finches (1:500), while PNNs were stained using Wisteria floribunda lectin (Sigma L1516; 1:1000).
+ Open protocol
+ Expand
6

Chondroitin Sulfate and Parvalbumin Labeling

Check if the same lab product or an alternative is used in the 5 most similar protocols
One well from each hemisphere of all birds was double-labeled for parvalbumin (PV) and chondroitin sulfate, one of the main components of the perineuronal nets, following a previously described protocol [22 (link), 37 (link), 41 (link)]. Briefly, sections were blocked in 5% Normal Goat Serum (NGS) diluted in Tris-buffered Saline (TBS) with 0.1% Triton-X-100 (TBST) for 30 minutes. They were incubated overnight at 4°C in a mixture of 2 primary antibodies diluted in TBST: a mouse monoclonal anti-chondroitin sulfate antibody (clone CS-56, 1:500 for Experiment 1 and 3 or 1:1000 for Experiment 2, C8035, Sigma Aldrich) specific for the glycosaminoglycan portion of the chondroitin sulfate proteoglycans that are the main components of the PNN and a polyclonal rabbit anti-parvalbumin antibody (1:1000; ab11427, Abcam). Sections were then incubated at room temperature in a mixture of secondary antibodies diluted in TBST. A goat anti-mouse IgG coupled with Alexa 488 (green, 1:100, Invitrogen) was used to visualize PNN staining and a goat anti-rabbit IgG coupled with Alexa 546 (red, 1:200, Invitrogen) was used to visualize PV cells. Finally, sections were mounted on slides using TBS with gelatin and coverslipped with Vectashield containing DAPI (H-1500, Vector laboratories) to confirm that PNN that were not surrounding PV-positive cells were localized around a cell nucleus.
+ Open protocol
+ Expand
7

Dual Immunostaining for Parvalbumin and Chondroitin Sulfate

Check if the same lab product or an alternative is used in the 5 most similar protocols
The telencephalic tissue from the 3rd series of sections was then simultaneously immunostained for PV and chondroitin sulfate to label perineuronal nets (PNN) as described previously (Cornez et al., 2015 (link), 2017b (link), 2018b (link)) to obtain an additional measure of HVC plasticity (van 't Spijker and Kwok, 2017 (link)). Sections were rinsed 3 × 5 min in TBS and incubated in blocking solution made of 5% NGS and 0.1% Triton X-100 in TBS. Sections were then incubated overnight in a mixture of two primary antibodies including a polyclonal rabbit raised against PV (Abcam ab11427; 1:1000 in TBS-T 0.1% Triton X-100) and a monoclonal mouse anti-chondroitin sulfate antibody (1:500 in TBS-T 0.1% Triton X-100, Sigma-Aldrich C8035) for 48 h at 4°C on a rotating shaker. On the next day, sections were then washed 3 × 5 min in TBS and incubated for 2 h at room temperature on a rotating shaker in a cocktail of secondary fluorescent antibodies containing goat anti-mouse Alexa Fluor 488 (1:100, Invitrogen) and goat anti-rabbit Alexa Fluor 546 (1:200, Invitrogen). Sections were rinsed 3 × 5 min in TBS and then mounted on glass slides. Sections were dried and coverslipped with Vectashield mounting medium containing 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI) to label all cell nuclei.
+ Open protocol
+ Expand
8

Immunocytochemical Analysis of NG2 in Neu7 Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
Immunocytochemistry was used to assess NG2 levels in Neu7 cells. Neu7 cells were seeded into eight-well chamber slides at 2000 cells per well in Neu7 cell culture medium and incubated overnight at 37 °C and 5% CO2/90% humidity. Medium was removed, cells were washed with 1× PBS, fixed with 4% PFA and nonspecific staining was blocked as above. An antibody against NG2 (AB5320, 1:200; Merck Millipore, Darmstadt, Germany), GFAP (Z0334; 1:200; DAKO, Glostrup, Denmark) or CS-56 (C8035; 1:200; Sigma-Aldrich ((Dublin, Ireland) was added. After 24 h of primary antibody incubation, the Neu7 cells were washed with 1× PBS three times (5 min/wash). The Neu7 cells were then incubated with secondary antibodies for 2 h at room temperature, washed and cell nuclei stained as above. Cells were imaged using confocal microscopy.
+ Open protocol
+ Expand
9

Immunocytochemistry for MMP-2 and O4 in Oligodendrocytes

Check if the same lab product or an alternative is used in the 5 most similar protocols
For MMP-2/O4 staining, OPCs were plated onto 24-well coverslips that were precoated with PLL, 1 μg/mL laminin, and 2 μg/mL aggrecan and incubated with vehicle or 2.5 μM ISP for 2 days at 37 °C. Cultured OPC or OL cells were fixed in 4% PFA and followed by blocking in PBST solution (10% normal goat serum and 0.2% Triton–X100 in PBS). Diluted primary antibodies were incubated with samples overnight at 4 °C and followed by appropriate secondary antibody goat anti-mouse or anti-rabbit IgM or IgG conjugated with Alexa Fluor 488 or 594 (1:500, Invitrogen). The following primary antibodies were used: PDGFRα (ab65258, Abcam, 1:250), O4 (Hybridoma Core, Cleveland Clinic), MBP (SMI-99P, Covance, 1:300), MMP-2 (Ab19167, Millipore, 1:200), NG2 (AB5320, Millipore, 1:250), and CS56 (C8035, Sigma, 1:250). Cells were mounted with Vecta Shield mounting medium with DAPI (Vector Laboratories).
+ Open protocol
+ Expand
10

Heparitinase Digestion of Cell HS Chains

Check if the same lab product or an alternative is used in the 5 most similar protocols
Heparitinase treatments were performed for the digestion of B6FS cell HS chains, as previously described (24 (link),26 (link)). In brief, cells seeded in 24-well plates were serum-starved for 24 h and then treated with heparitinase (0.001 U/ml) for 24 and 48 h in 0% FBS medium. The cell extracts treated with heparitinase were electrophoresed on 8% polyacrylamide Tris/glycine gels and transferred onto nitrocellulose membranes. After blocking, the membranes were incubated for 1 h at room temperature with primary antibodies [mouse anti-heparitinase stubs antibody (clone 3G10; 1:500; CF500913; Seigakagu, Tokyo, Japan); goat anti-actin (1:200; sc-1616; Santa Cruz Biotechnology, Inc.) and mouse anti-CSA (1:200; C8035; Sigma)]. The immune complexes were detected following incubation with peroxidase-conjugated anti-goat or anti-mouse antibody, 1:4,000 or 1:2,000, respectively, with the SuperSignal West Pico Chemiluminescent substrate (Pierce Biotechnology, Inc.)
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!