Organoids were cultured in 4-well chamber slides (BD Biosciences), fixed in 4% PFA for 30 min, washed with PBS + 4% NaCl and blocked and permeabilized in blocking buffer (5% FBS, 0.2% BSA, 0.3% Triton X-100 in PBS) for 30 min. The following primary antibodies were applied at 4 °C overnight in blocking buffer: rat anti-vimentin (MAB2105, R&D Systems), rabbit anti-lumican (ab168348, Abcam), rabbit anti-active caspase-3 (AF835, R&D Systems), rabbit anti-Ki67 (Abcam, ab16667), rabbit anti-mucin2 (Santa-Cruz Biotechnology, sc-15334). After washing in PBS containing 0.3% Triton X-100 and 4% NaCl and overnight incubation with the secondary antibodies (Alexa Fluor 488 and 594, Thermo Fisher), the organoids were mounted in mounting medium containing DAPI (Thermo Fisher) and imaged with an Olympus FV500 confocal microscope.
Ab168348
Manufactured by Abcam
Sourced in United Kingdom
Ab168348 is a rabbit monoclonal antibody targeted against a specific protein. This antibody can be used for various research applications, such as Western blotting and immunohistochemistry. The core function of this product is to provide a tool for the detection and study of the target protein.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Mounting medium, by Thermo Fisher Scientific (1 mentions)
Fv500 confocal microscope, by Olympus (1 mentions)
Ab16667, by Santa Cruz Biotechnology (1 mentions)
Sc 15334, by Santa Cruz Biotechnology (1 mentions)
4 well chamber slide, by BD (1 mentions)
Mab2105, by R&D Systems (1 mentions)
Af835, by R&D Systems (1 mentions)
Ab228549, by Abcam (1 mentions)
Ts2 fc, by Nikon (1 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Ab150077, by Abcam (1 mentions)
Ab150115, by Abcam (1 mentions)
Ab6310, by Abcam (1 mentions)
Ab81032, by Abcam (1 mentions)
Ab27478, by Abcam (1 mentions)
Ab6308, by Abcam (1 mentions)
Vector ba 1000, by Vector Laboratories (1 mentions)
3 protocols using ab168348
1
Immunofluorescence Staining of Organoids
2
Cytoskeletal Organization and Colocalization
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HVSMCs (6000/well) were seeded into six-well plates with coverslip until reaching 80% confluence. After treatment, cells were fixed with 10% formalin, and permeabilized by 0.2% Triton X-100 (G5060; Servicebio, China). Next, cells were reacted with Alexa Fluor® 647 Anti-Tubulin antibody (ab195884; Abcam, UK) at 4℃ overnight. A fluorescence microscope (200×, Ts2-FC; Nikon, Japan) was used to capture and observe the staining results.
For colocalization, cells were incubated with rabbit anti-LUM (ab168348, 1:250; Abcam) and mouse anti-APOB (GTX60445, 1:200; GeneTex) antibodies, followed by successive culture with secondary antibodies, Alexa Fluor® 488 (ab150077; Abcam) and Alexa Fluor® 647 (ab150115; Abcam). Nuclei were labeled with DAPI (ab228549; Abcam). Protein images were captured using Zeiss LSM 710 confocal microscope (Carl Zeiss AG, Oberkochen, Germany).
For colocalization, cells were incubated with rabbit anti-LUM (ab168348, 1:250; Abcam) and mouse anti-APOB (GTX60445, 1:200; GeneTex) antibodies, followed by successive culture with secondary antibodies, Alexa Fluor® 488 (ab150077; Abcam) and Alexa Fluor® 647 (ab150115; Abcam). Nuclei were labeled with DAPI (ab228549; Abcam). Protein images were captured using Zeiss LSM 710 confocal microscope (Carl Zeiss AG, Oberkochen, Germany).
3
Quantitative Immunohistochemical Analysis of Carotid Plaque
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Carotid plaque sections were immunohistochemically stained using primary antibodies against fibromodulin (kindly provided by D. Heinegård, Lund University, Sweden), lumican (ab168348; Abcam, Cambridge, UK), Glycophorin A (M0819, Dako Sweden, Stockholm, Sweden), collagen I and collagen III (ab6308 and ab6310 respectively; Abcam, Cambridge, UK). A biotinylated goat anti-rabbit IgG (Vector BA-1000, Vector Laboratories Inc, Burlingame, CA, USA) or rabbit F(ab')2 anti-mouse IgG (ab98668, Abcam, Cambridge, UK) was used as secondary antibodies. In addition, immunohistochemical stains for smooth muscle α-actin and CD68) were performed as previously described. 23, (link)24 (link) Isotype control antibodies were used in concentrations corresponding to that of each primary antibody (ab27478 and ab81032from rabbit and mouse, respectively; Abcam, Cambridge, UK) -representative images are shown in Supplementary Figure I. Immunoreactivity was quantified blindly using the imaging software program BioPix iQ version 2.3.1 (Biopix Ab, Gothenburg, Sweden). Residual media was not included during image analysis.
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