PDOs isolated from Matrigel using Cell Recovery Solution (354,253; Corning) were embedded in iPGell (PG20-1; GenoStaff, Tokyo, Japan) without damaging the 3D structures according to the manufacturer’s protocol. PDOs were then fixed with 4% paraformaldehyde and used to create paraffin-embedded blocks. Paraffin-embedded blocks of PDOs and PDAC tissues were sectioned (4 μm) and subjected to standard hematoxylin and eosin (H&E) staining and immunostaining. The following primary antibodies were used: E-cadherin (ab15148; abcam, Cambridge, UK, 1:30), Actin (ab130935; abcam, 1:100), CK19 (sc25724; Santa Cruz Biotechnology, Inc., Dallas, TX, USA, 1:50), αSMA (M0851; Dako, Santa Clara, CA, USA, 1:200), and RSPO3 (17,193–1-AP; proteintech, Rosemont, IL, USA, 1:100). The secondary antibodies were EnVision + System-HRP Labelled Polymer (K4003; Dako) and Alexa Fluor 488 anti-rabbit (A11034; ThermoFisher) and 546 anti-mouse (A11030; ThermoFisher). Nuclei were counterstained with hematoxylin or 4′,6-diamidino-2-phenylindole (DAPI; Dojindo, Kumamoto, Japan). For immunohistochemistry, staining was developed with 3,3′-diaminobenzidine substrate chromogen (11,209-1A; Kanto Kagaku, Tokyo, Japan) Bright field images were acquired using BZ-X700. The stain-positive area was quantified using HybridCellCount software module of BZ-X Analyzer (Keyence).
Ipgell
Manufactured by Genostaff
Sourced in Japan
IPGell is a lab equipment product used for isoelectric focusing, a technique in proteomics and biochemistry. It facilitates the separation of proteins based on their isoelectric point.
Automatically generated - may contain errors
Lab products found in correlation
Cell recovery solution, by Corning (4 mentions)
Cell recovery solution, by BD (4 mentions)
96 well round bottom ultra low attachment microplates, by Corning (2 mentions)
Dako real envision detection system, by Agilent Technologies (2 mentions)
Histofine kit, by Nichirei Biosciences (2 mentions)
α sma, by Agilent Technologies (1 mentions)
Envision system hrp labelled polymer, by Agilent Technologies (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (1 mentions)
E cadherin, by Abcam (1 mentions)
Bz x analyzer, by Keyence (1 mentions)
Hybrid cell count software, by Keyence (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Nacalai Tesque (1 mentions)
Anti cd44, by Cell Signaling Technology (1 mentions)
Anti cd133, by Abnova (1 mentions)
Bz 9000, by Keyence (1 mentions)
Bovine serum albumin (bsa), by Nacalai Tesque (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Nacalai Tesque (1 mentions)
Triton x 100, by Merck Group (1 mentions)
34 protocols using ipgell
1
Establishing 3D Organoid Culture and Immunohistochemistry
2
Hypoxia Detection in Pseudoislet Aggregates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hypoxic cells in the pseudo-islets were detected by a Hypoxyprobe-1 Kit (Hypoxyprobe, Burlington, MA). After 7 days of cultivation, the aggregates in the microwells were incubated for 2 h in the presence of 200 μM pimonidazole hydrochloride (Hypoxyprobe-1). Then, the aggregates were collected, embedded in iPGell (GenoStaff, Tokyo, Japan), and fixed with 4% paraformaldehyde (PFA) for 4 h according to the manufacturer's instructions. The gels containing aggregates were embedded in paraffin and sectioned at 5 μm. The sections were incubated with mouse monoclonal anti-pimonidazole antibody overnight at 4 °C. Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Thermo Fisher Scientific) was used as the secondary antibody. The sections were mounted with ProLong Gold Antifade Reagent with DAPI (Thermo Fisher Scientific). The images were taken with a fluorescence microscope (Olympus BX51).
3
Optimizing pMLKL Staining and PLA Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cell blocks were prepared for optimization and validation of pMLKL staining and in situ PLA assay. HT-29 cells were solidified using iPGell (Genostaff, Tokyo, Japan) according to the manufacturer’s instructions. Briefly, cells were collected and fixed with 10% neutral-buffered formalin for 10 min. Solutions A and B were added to solidify the cells. The clot was gently removed from the tube and transferred to a tissue cassette. Subsequently, a tissue cassette was soaked into ethanol and xylene and finally embedded in paraffin.
4
DCIS Organoid Generation and Fixation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We seeded 1 × 105 DCIS cells onto 150 μl of Matrigel coating a 24-well plate, followed by culture with the DMEM/ F12 medium with GlutaMAX supplement containing 5% FBS overnight (Figs 2C and S2A ). Floating dead cells and media were removed the next day and viable cells attached to the Matrigel were covered with 50 μl of Matrigel and overlaid with media containing 5% FBS. The culture medium was changed every second day and the cells were cultured for 5 d. The Matrigel was mechanically destroyed and the organoids were collected into a 1.5-ml tube prior to being jellied using iPGell (GenoStaff) and fixed in formalin solution.
5
Immunohistochemical Analysis of 3D-Organoids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunohistochemical analysis, 3D-organoids were embedded in iPGell (Geno Staff) and fixed overnight in 4% paraformaldehyde (Nacalai Tesque). Tumor specimens were isolated and fixed overnight in 4% paraformaldehyde (Nacalai Tesque), embedded in paraffin and sectioned at a thickness of 3 or 4 μm. Sections were then deparaffinized, rehydrated, and stained with hematoxylin and eosin (HE). For immunohistochemical analyses, standard IHC procedures were performed in a BOND-RX automated immunostaining machine (Leica) according to the manufacturer’s instructions using anti-CD44 (1:600, Cell Signaling Technologies) and anti-CD133 (1:200, Abnova) antibodies. Images of the stained slides were captured and analyzed using an Aperio ImageScope (Leica, 20x objective lens) or inverted microscope systems (Olympus IX83 or Keyence BZ9000, 10x or 20x objective lenses) with the built-in software and ImageJ.
6
Gastric Organoid Culture and Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The organoid cultures were prepared from gastric epithelial cells and T1-T3 cells, as previously described23 (link),31 . Cells were seeded at a density of 1.3 × 105 cells per 3-cm dish on day 0 and analyzed on day 5. The cell suspension was recovered from Matrigel’s basement membrane matrix using Cell Recovery Solution (Corning). The cell suspension was hardened in jelly form with iPGell (GenoStaff) and fixed in 4% paraformaldehyde, paraffin embedded and sectioned at 5- μm thickness. The sections were stained with Haemotoxylin and Eosin.
7
Organoid Cell Block Generation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Organoid pellets were prepared by dissolving Matrigel with cell recovery solution (Corning) at 4°C for approximately 30 min. To create an organoid cell block, the prepared organoid pellet was solidified using iPGell (Genostaff), according to the manufacturer's instructions. Immunohistochemistry of each marker was performed using organoid cell blocks and organoid‐derived mouse xenograft. The detailed method is described in the Supplementary Methods .
8
Spheroid Formation Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spheroid formation ability was assessed as previously reported [30 (link)]. Cells (1 × 105) were seeded in 96-well round-bottom ultra-low attachment microplates (Corning, Inc., Corning, NY, USA) and incubated for 3 days. Spherical colonies fixed with iPGell (Genostaff, Tokyo, Japan) were prepared for paraffin sections, which were stained with H&E.
9
Immunocytochemical Staining of Org-HYDROX Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To perform the immunocytochemistry, Org-HYDROX washed with PBS were embedded in iPGell (GenoStaff) according to the manufacturer’s instructions, and then fixed with 4% paraformaldehyde for 15 min. After blocking the cells with PBS containing 2% bovine serum albumin (Nacalai Tesque) and 2% Triton X-100 (Merck) for 30 min at room temperature, the cells were incubated with the blocking buffer containing a primary antibody (described in Table S4 ) for 3 h at room temperature, and finally with the blocking buffer containing a secondary antibody (described in Table S4 ) for 1 h at room temperature. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (Nacalai Tesque). Images were acquired with fluorescence microscope (Biozero BZ-X800; KEYENCE).
10
FISH Analysis of CIC Rearrangement
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
FISH analysis was performed on 4-µm-thick sections. A solid cell pellet was prepared from adherent tumour cells by iPGell (Geno Staff). Break-apart probes were used for CIC (custom-made probe; Chromosome Science Labo) genes as previously described12 (link). FISH images were obtained using the Metafer Slide Scanning Platform (MetaSystems). The presence of split 5′ and 3′ signals or isolated 5′ signals in more than 20% of tumour cells was considered positive for CIC rearrangement.
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
Revolutionizing how scientists
search and build protocols!