Human NP cells grown on cover glass underwent fixation with 4% formalin (20 min) at ambient, permeabilization with 0.1% Triton X-100 and 0.2% Tween-20 in PBS (40 min at ambient), blocking with 2% goat serum (Invitrogen; 1 h), and incubation with anti-collagen-II (1:200; Abcam, Ab34712), anti-Aggrecan (1:500; Abcam, Ab5790), anti-MMP13 (1:50; Abcam, Ab21624), and anti-ADAMT-5 (1:1000; Millipore, MAB4401) primary antibodies, respectively. After washing, the samples further underwent incubation with fluorescein-conjugated secondary antibodies. Images were captured under a fluorescence microscope (Leica).
Ab21624
Manufactured by Merck Group
Ab21624 is a laboratory equipment product. It is designed for use in scientific research and analysis. The core function of this product is to facilitate specific tasks within a laboratory setting. Further details about its intended use or application are not available.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence microscope, by Leica (3 mentions)
Ab5790, by Abcam (3 mentions)
Goat serum, by Thermo Fisher Scientific (2 mentions)
Ab34712, by Abcam (2 mentions)
Mab4401, by Merck Group (2 mentions)
Mab4304, by Abcam (2 mentions)
Af1924, by Abcam (1 mentions)
Vectashield h 1200 mounting media, by Vector Laboratories (1 mentions)
Af2085, by R&D Systems (1 mentions)
Mab4401, by Abcam (1 mentions)
Ab5603, by Abcam (1 mentions)
Las 4000 luminescence image analyzer, by Fujifilm (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Ab77013, by Abcam (1 mentions)
Ab19857, by Abcam (1 mentions)
Ab47003, by Abcam (1 mentions)
Ab8295, by Abcam (1 mentions)
Ab91196, by Abcam (1 mentions)
6 protocols using ab21624
1
Immunofluorescence Characterization of Chondrocytes
2
Pluripotent Stem Cell Characterization
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Human ESCs and iPSCs were cultured on cover glass and fixed by incubating with 4% paraformaldehyde for 20 min at room temperature (RT). Fixed cells were then washed with PBS and permeabilized using a non-ionic detergent (0.1% Triton X-100 and 0.2% Tween-20) in PBS for 40 min at RT. Permeabilized cells were blocked by incubating with 2% goat serum (Invitrogen) for 1 h, washed with PBS containing 0.01% Tween-20 (PBST), and incubated with primary antibody. Primary antibodies used included anti-SOX17 (1:200; R&D Systems; AF1924), anti-PAX6 (1:500; Abcam; Ab5790), anti-NANOG (1:50; Abcam; Ab21624), anti-OCT4 (1:1000; Millipore; MAB4401), and anti-Brachyury (1:50; R&D Systems; AF2085). Cells were then washed with PBST and incubated with the appropriate fluorescein-conjugated secondary antibody. Stained samples were mounted using Vectashield H-1200 mounting media (Vector Laboratories), and images were captured using a fluorescence microscope (Leica). Positive signals in IF images were processed and counted using Image J with a consistent intensity threshold.
3
Western Blot Analysis of Stem Cell Markers
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Protein was extracted with RIPA buffer and quantified using a BCA protein assay kit (Pierce). For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 20 μg of protein from each sample was resolved on 10% gels and electro-transferred to a nitrocellulose membrane. After blocking non-specific binding by incubating in 3% non-fat milk in PBST for 1 h, at RT, membranes were incubated at 4 °C overnight with diluted primary antibody. The primary antibodies used were anti-OCT4 (1:1000; Millipore; MAB4401), anti-NANOG (1:1000; Abcam; Ab21624), anti-SOX2(1:2000; Millipore; AB5603), anti-AGO2 (1:1000; Abcam; ab57113), anti-ESRP1 (Novus; NBP1-82201), and mouse anti-β-actin (1:5000; Sigma; A5441). After washing with PBST, membranes were incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h, at RT. Signals were developed using Supersignal West Femto Substrate (Pierce) and recorded with an LAS-4000 luminescence image analyzer (Fujifilm). Full scans of the western blots shown in Fig. 5 are provided in Supplementary Fig. 11 .
4
Chondrocyte Extracellular Matrix Analysis
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Human NP cells grown on cover glass underwent fixation with 4% formalin (20 min) at ambient, permeabilization with 0.1% Triton X-100 and 0.2% Tween-20 in PBS (40 min at ambient), blocking with 2% goat serum (Invitrogen; 1 h) and incubation with anti-collagen-II (1:200; Abcam, Ab34712), anti-Aggrecan (1:500; Abcam, Ab5790), anti-MMP13 (1:50; Abcam, Ab21624), and anti-ADAMT-5 (1:1000; Millipore, MAB4401) primary antibodies, respectively. After washing, the samples further underwent incubation with fluorescein-conjugated secondary antibodies. Images were captured under a fluorescence microscope (Leica). Relative fluorescence intensity was calculated with Image J.
5
Immunocytochemical Staining of Stem Cell Markers
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The immunocytochemical staining were performed according to the protocol described previously [4 (link),38 (link)]. Briefly, cells seeded on slides or SIS patches were fixed with 4% paraformaldehyde (4% PFA) and permeabilized in 0.4% Triton X-100 (Sigma). After blocking with 10% normal goat serum (Vector Laboratories), the cells were then incubated with primary antibodies against octamer-binding transcription factor 4 (OCT4, Abcam, ab19857, 1:100), nanog homeobox (NANOG, Abcam, ab21624, 1:100), SSEA4 (Millipore, MAB4304,1:300), MESP1 (Abcam, ab77013, 1:100), ISL1 (Abcam, ab178400, 1:200), and NKX2-5 (Abcam, ab91196, 1:200), cTnT (Abcam, ab8295, 1:400), cTnI (Abcam, ab47003, 1:400), α-actinin (Abcam, ab9465, 1:400), or GATA4 (Stem Cell, SC-25310, 1:100) in 4 °C overnight and detected by DyLight 488- or 549-conjugated secondary antibodies. Nuclei were stained with Hoechst33258 (Sigma). Zeiss fluorescence microscope was used for slide observation and image capture.
6
Characterizing Pluripotent & Neural Stem Cells
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For alkaline phosphatase (AP) staining, hESCs were fixed with 4% paraformaldehyde (PFA) in PBS for 40 s, rinsed once with PBS and stained using a leukocyte alkaline phosphatase kit (Sigma-Aldrich, St Louis, MO, USA) according to the manufacturer's protocol. For immunofluorescent staining, cells were fixed with 4% PFA for 15 min. The fixed cells were washed with PBS and incubated in 0.2% Triton X-100 for 20 min, then washed with PBS and incubated in blocking buffer containing 20% FBS, 10% Glycerol, 100 nM Glycine and 0.1% Triton X-100 for 30 min at room temperature. The cells were then incubated with primary antibodies against Oct4 (Abcam, ab19857), SSEA-4 (Millipore, MAB4304), Nanog (Abcam, ab21624), Nestin (Millipore, MAB5326), Sox2 (Abcam, ab15830), Sox1 (Abcam, ab22572), Pax6 (DSHB, P3U1), Musashi1 (Abcam, ab21628), Tuj1 (Covance, PRB-435P), GFAP (Invitrogen, 13-0300) or O4 (R&D, MAB1326) overnight at 4 °C. The following day cells were washed with PBS and incubated in the appropriate secondary antibodies conjugated to Alexa Fluor 546 or Alexa Fluor 488 (Invitrogen) for one hour at room temperature. Nuclei were counterstained with Hoechst 33342 for 10 min. Images were taken with an Olympus IX71 inverted fluorescent microscope or an Olympus FV10i confocal microscope.
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