DF, mesenchymal stem cells (MSC), and lung carcinoma epithelial cells were tested for expression of epithelial (keratin) and mesenchymal (vimentin) markers. Furthermore, DF cultured on ECMs were tested for the expression of αvβ3 integrin. Cells were fixed in 24-well plates with formaldehyde (0006450323F1, Bio-Lab, Ltd., Jerusalem, Israel). Endogenous peroxidase activity was quenched in PBS-H2O2 1% (3 mL H2O2 3% + PBSx1 FLORIS). Cells were blocked using goat serum (04-009-1A, Biological Industries, Kibbutz Beit-Haemek, Israel) and incubated with a primary antibody (rabbit anti-Vimentin, rabbit anti-keratin, or rabbit anti-αvβ3 integrin; details in Table 3 ), at 4 °C overnight. Cells were washed and incubated with horseradish peroxidase-labeled polymer conjugated to a secondary antibody (ZUC053006 Zytomed, Berlin, Germany), washed, and developed with the 3-amino-9-ethylcarbazole (AEC) substrate buffer (ACG500, ScyTek Laboratories, Logan, UT, USA). Cell nuclei were stained with hematoxylin (MFCD00147088, Merck, Darmstadt, Germany).
Acg500
Manufactured by ScyTek Laboratories
Sourced in United States
The ACG500 is a laboratory centrifuge designed for general-purpose applications. It provides a reliable and efficient method for separating a wide range of sample types, including biological fluids and suspensions. The ACG500 features a robust construction, user-friendly controls, and can accommodate a variety of sample containers.
Automatically generated - may contain errors
Lab products found in correlation
Cytoseal 60, by Thermo Fisher Scientific (1 mentions)
Avidin d solution, by Vector Laboratories (1 mentions)
Ba 1000, by Vector Laboratories (1 mentions)
Chondroitinase abc, by Merck Group (1 mentions)
Ab1031, by Merck Group (1 mentions)
Pk 6100, by Vector Laboratories (1 mentions)
Blocking reagent, by Thermo Fisher Scientific (1 mentions)
Hmm500, by ScyTek Laboratories (1 mentions)
Anti muc5ac antibody, by Abcam (1 mentions)
3 protocols using acg500
1
MSC and Carcinoma Cells Characterization
2
Immunohistochemical Detection of Aggrecan in IVD
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Detection of aggrecan was performed on deparaffinized coronal IVD tissue sections of 4‐μm thickness. The sections were pretreated with chondroitinase ABC (0.25 U/mL; C3667 Millipore Sigma) at 37°C for 1‐h to unmask epitopes on aggrecan. Endogenous peroxidase activity was eliminated by the treatment with 3% hydrogen peroxide, and then incubated 15 min with Avidin D solution and 15 min with biotin solution (SP‐2001, Vector laboratories). Permeabilization and blocking was done with 10% goat serum, 1% BSA, and 0.25% triton x‐100 in PBS for 30 min. Primary antibody against aggrecan (1:200; AB1031, Millipore Sigma) was applied at 4°C overnight, then thoroughly washed with washing buffer (0.05% Tween20 in PBS). Biotinylated goat anti‐rabbit secondary antibody (1:200; BA‐1000, Vector Laboratories) was applied for 30 min and the sections were washed again in washing buffer, followed by the avidin‐biotin amplification (PK‐6100, Vector Laboratories) for 30 min. AEC substrate/chromogen KIT (ACG500, ScyTek Laboratories) was used for 10 min. Hematoxylin was used as a counterstain. All mounted slides with mounting medium Cytoseal 60 (8310‐4, Thermo Fisher Scientific) were visualized on a Nikon Eclipse E800 microscope. Mean aggrecan intensity was calculated on image J using an established protocol.
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3
Immunohistochemical Analysis of IL-20 and MUC5AC in Eye Tissues
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Eyes tissues were fixed in 4% paraformaldehyde solution and were proceed with dehydration and embedding according to common methods by Human Biobank in National Cheng Kung University Hospital. Paraffin-embedded tissue sections (4 μm) were deparaffinized and rehydrated and subjected to heat-induced antigen retrieval by citrate buffer (pH 6.4) for 95 °C 30 min. Nonspecific binding was blocked by treatment with blocking reagent (Thermo Fisher Scientific, Cat# 003118). The sections were incubated with anti-IL-20 antibody (7E) or anti-MUC5AC antibody (Abcam, Cat# ab3649) at 4 °C overnight. 7E were prepared as previously described [37 (link), 38 (link)]. The next day, the sections were washed with PBS and incubated with the secondary antibody for 1 h. The reaction was detected using AEC chromogen stain (ScyTek Laboratories, Cat# ACG500), and the nuclei were counterstained with hematoxylin (ScyTek Laboratories, Cat# HMM500). Pictures were taken under an inverted fluorescence microscope IX71 (Olympus).
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