Liver tumor organoids were fixed in 4% paraformaldehyde (Tech and Innovation, Chuncheon, Republic of Korea) for 1 h and incubated overnight at 4 °C in 30% sucrose phosphate buffer. Thereafter, the liver organoids were embedded in FSC-22 frozen section medium (Leica Biosystems, Wetzlar, Germany) and cut into 10 µm-thick sections using a cryostat at −20 °C. The sections were then permeabilized using 1% Triton X100 in phosphate-buffered saline (PBS) for 10 min, and blocked using blocking buffer (5% bovine serum albumin in PBS containing 0.2% Triton X100) for 30 min. The sections were then incubated overnight at 4 °C with primary antibodies against CK8 (sc-8020; Santa Cruz Biotechnology), EMR1 (sc-377009; Santa Cruz Biotechnology), and cleaved caspase-3 (CC3, MAB835; R&D Systems) in blocking buffer. For fluorescence labeling, the sections were incubated with Alexa Fluor 488- or Alexa Fluor Plus 647-conjugated secondary antibody (1:100; Invitrogen, Carlsbad, CA, USA) for 1 h at 25 °C. Nuclear staining was conducted using DAPI (4′,6-diamidino-2-phenylindole; Sigma-Aldrich, St. Louis, MO, USA), and samples were mounted using DPX Mounting Medium (Electron Microscopy Sciences, Hatfield, PA, USA). The sections were then observed and imaged under a confocal microscope (LSM800; Zeiss, Oberkochen, Germany).
Sc 8020
Manufactured by Santa Cruz Biotechnology
The Sc-8020 is a laboratory centrifuge designed for general-purpose applications. It features a compact and durable construction, with a maximum speed of 6,000 rpm and a maximum capacity of 4 x 250 mL. The Sc-8020 is suitable for a variety of sample preparation and separation tasks in research and diagnostic laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Sc 377009, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor plus 647 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Dpx mounting medium, by Electron Microscopy Sciences (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Merck Group (1 mentions)
Ab735, by Abcam (1 mentions)
Ab223343, by Abcam (1 mentions)
Ab92320, by Abcam (1 mentions)
Fv3000 laser confocal microscope, by Olympus (1 mentions)
Ab7817, by Abcam (1 mentions)
A21206, by Thermo Fisher Scientific (1 mentions)
Bx35 microscope, by Olympus (1 mentions)
A31570, by Thermo Fisher Scientific (1 mentions)
Ab109536, by Abcam (1 mentions)
Ab209665, by Abcam (1 mentions)
Ab652, by Abcam (1 mentions)
Ab64064, by Abcam (1 mentions)
Nb100 105, by Novus Biologicals (1 mentions)
Cw0096, by CWBIO (1 mentions)
4 protocols using sc 8020
1
Immunofluorescence Analysis of Liver Tumor Organoids
2
Histology Staining of Organoid Samples
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The histology staining protocol followed the methods in the previous study [23 (link)]. Briefly, organoids were washed twice with cold PBS, fixed with 4% paraformaldehyde for 15 minutes, and then removed into the mold to dehydrate and embedded in paraffin. Sections were subjected to standard H&E, IHC, and IF staining. The following primary and secondary antibodies were used in this study: Cytokeratin 5 (CK5,1:200, EP1601Y, Invitrogen), Cytokeratin 8 (CK8, 1:200, sc-8020, Santa cruz), p63 (1:200, ab735, Abcam), Aquaporin 5 (AQP5, 1:400, ab92320, Abcam), alpha-smooth muscle actin (SMA,1:200, ab7817, Abcam), DOG-1 (GT205402, GeneTech), SOX-10 (GT221002, GeneTech), C-erbB-2 (HER-2, GT224502, GeneTech), Androgen receptor (AR, GM356202, GeneTech), MUC-4 (GT227502, GeneTech), PTP4A1(1:400, DF12458, Affinity), NEFL (1:50, ab223343, Abcam), Alexa Fluor 488 (1:300, A-21206, Invitrogen), Alexa Fluor 555 (1:300, A-31570, Invitrogen). Images were acquired with Olympus BX35 microscope and Olympus laser confocal microscope FV3000.
3
Western Blot Analysis of Stem Cell Markers
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Cell samples were lysed in RIPA buffer on ice for 20 min. The lysed cells were collected and centrifuged at 14,000 x g to remove cell debris. Protein concentrations were determined with the BCA Protein Assay Kit (P0009, Beyotime). Each sample containing 30 μg of total protein was separated by SDS-PAGE in a 10% gel and transferred onto PVDF membranes (EMD Millipore Corporation, US). After blockage with 5% nonfat dry milk in Tris-buffered saline with 1‰ Tween (TBST), the membranes were incubated overnight at 4 °C with primary antibodies against brachyury (ab209665, Abcam), GLUT-1 (ab652, Abcam), SHH (sc-365,112, Santa Cruz), CD24 (ab64064, Abcam), KRT-8 (sc-8020, Santa Cruz), HIF-1α (NB100–105, Novus), NOTCH1 (4380, CST), JAGGED1 (ab109536, Abcam), HES1 (11,988, CST), and β-actin (CW0096S, CW Biotech). After three washes with TBST, the membrane was incubated with horseradish peroxidase–conjugated secondary antibodies (Jackson). The protein expression level was determined by densitometric analysis and was normalized to the level of β-actin.
4
Multiplex Detection of SARS-CoV-2 and ACE2 RNA
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Tissue sections (4 µm thick) were cut from FFPE tissue blocks and mounted onto glass slides. Slide-tissue sections were baked at 70 °C for 1 h and subsequently soaked in xylene (Thermo Fisher Scientific) for 10 min × 3 rounds. Rehydration and HIER of tissue sections were performed in a similar manner as described above except that rehydrating washes were done for 180 s each. After HIER and cooling, slides were then washed twice with Milli-Q water (Millipore Sigma) subjected to a 10-min protease digestion at 40 °C with Protease III (322337, Bio-Techne) diluted to 1:20 in 1X PBS. Slides were then washed for 2 × 2 min Milli-Q water before a 15-min H2O2 block at 40 °C (322335, Bio-Techne). Slides were then washed for 2 × 2 min Milli-Q water before an overnight hybridization at 40 °C with probes against the human ACE2 mRNA (848151, Bio-Techne) or SARS-CoV-2 Spike mRNA (848561, Bio-Techne). Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for ACE2 mRNA probe targets (NEL744001KT, Akoya Biosciences). Slides were then processed as described above for IF IHC staining for anti-MUC5AC (Abcam ab212636), ACE2 (Abcam ab15348), and/or cytokeratin 8 (Santa Cruz sc-8020) staining. Fluorescent images were acquired and processed as detailed above.
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