Before treatment with 5 μg/ml Cetuximab, HCT116 and HT29 cells were cultured in 8-well chamber slides (Matsunami glass, Osaka, Japan) for 1 day. After washing with PBS twice, the cells were fixed for 20 min with 4% paraformaldehyde (Merck KGaA) and permeabilized for 15 min with 0.05% Triton-X 100 (Merck KGaA). The samples were blocked for 30 min in 5% bovine serum albumin (BSA) (Merck KGaA) in PBS and then incubated overnight at 4 °C with EGFR antibody (100-fold dilution) in PBS containing 10% FBS and 1% BSA. After washing with PBS 4 times, the samples were incubated for 30 min with secondary antibody conjugated with Alexa Fluor 488 (Abcam) (1000-fold dilution) in PBS containing 10% FBS and 1% BSA. After washing with PBS 4 times, the slides were mounted in ProLong Gold antifade reagent with 4,6-diamidino-2-phenylindole (Thermo Fisher Scientific) and analyzed with a LSM710 confocal microscope (Carl Zeiss, Oberkochen, Germany).
8 well chamber slides
Manufactured by Matsunami
Sourced in Japan
The 8-well chamber slides are a laboratory equipment designed for cell culture applications. Each slide features eight individual chambers, providing a convenient platform for conducting multiple experiments or observations simultaneously. The chambers are made of durable materials and are suited for a variety of cell types and experimental setups.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Mmw 001, by Matsunami (2 mentions)
Bz 9000, by Keyence (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Alexa fluor 488, by Abcam (1 mentions)
Prolong gold antifade reagent with 4 6 diamidino 2 phenylindole, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Softworx, by GE Healthcare (1 mentions)
Rat collagen 1, by Corning (1 mentions)
Nucleolus bright red, by Dojindo Laboratories (1 mentions)
Deltavision elite, by GE Healthcare (1 mentions)
Blocking one solution, by Nacalai Tesque (1 mentions)
Anti collagen 4, by Abcam (1 mentions)
Anti collagen 1, by Abcam (1 mentions)
Anti laminin α2, by Merck Group (1 mentions)
Anti collagen 6, by Abcam (1 mentions)
Dapi containing mounting medium, by Vector Laboratories (1 mentions)
7 protocols using 8 well chamber slides
1
EGFR Localization in Colon Cancer Cells
2
Immunofluorescence Staining of Infected Cells
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Cells were plated in 8-well chamber slides (Matsunami, Osaka, Japan) coated with rat collagen I (Corning, Corning, NY). Infected or transfected cells were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS; Nacalai Tesque, Japan) for 10 min and then permeabilized with 0.5% Triton X-100 in PBS for 5 min. The cells were blocked with Blocking One solution (Nacalai Tesque) for 30 min, followed by incubation with primary antibodies overnight at 4°C and secondary antibodies for 1 h at room temperature. For nuclei and rRNA staining, the cells were treated with Hoechst 33342 (Thermo Fisher Scientific) and Nucleolus Bright Red (Dojindo, Japan), respectively, for 10 min. Section images were recorded using DeltaVision Elite (GE Healthcare) with a 60× oil objective and then deconvolved and projected using the Quick Projection tool by softWoRx (GE Healthcare).
3
Proximity Ligation Assay for NHEKs
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NHEKs were seeded in 8-well chamber slides (MATSUNAMI, Osaka, Japan) and incubated without stimulation or with LL-37 alone or in combination with poly(I:C) for 1 h at 4 °C to prevent internalization. Cells were washed with cold PBS and fixed in cold 4% paraformaldehyde (PFA). After blocking according to the manufacturer’s instructions (Sigma-Aldrich), cells were incubated with two primary antibodies. Secondary antibodies conjugated with oligonucleotides were added, then ligation and amplification were performed to generate a fluorescent signal in the region where the antigen recognized by the two primary antibodies was present below 40 nm. Fluorescent PLA signals were detected and photographed with a laser scanning confocal microscope (LSM700; ZEISS, Tokyo, Japan). PLA signal counts were measured with ImageJ software (Fiji; version 2.1.0; National Institutes of Health, Bethesda, MD, USA).
4
Immunohistochemistry and Immunocytochemistry Analysis
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Immunohistochemistry was performed using the cryosections described above. For immunocytochemistry, primary satellite cells were cultured on 8-well chamber slides (MATSUNAMI) coated with Matrigel. Tissue sections or cells were fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde for 10 min at room temperature, and then permeabilized with PBS containing 0.2% Triton X-100 (Sigma–Aldrich) for 15 min at room temperature. After blocking with Power Block Universal Blocking Reagent (BioGenex) or a M.O.M. kit (Vector Laboratories), the fixed cells were incubated with primary antibodies overnight at 4 °C. After washing, bound primary antibodies were labeled with fluorescence-conjugated secondary antibodies for 1 h at room temperature. The immunostained samples were mounted with Mounting medium containing DAPI (Vector Laboratories). The primary and secondary antibodies were as follows: anti-laminin α2 (Sigma–Aldrich), anti-Pax7 (SantaCruz), anti-collagen I (Abcam), anti-collagen IV (Abcam), anti-collagen VI (Abcam) and mouse/rabbit/rat IgG-Alexa488 or -Alexa594 (Life Technologies).
5
Immunocytochemical Detection of AFP and Albumin in 201B7 Cells
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201B7 cells were cultured on 8-well chamber slides (Matsunami, Kishiwada, Japan) coated with MatrigelTM. After culture, the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) at 4°C for 10 min. The fixed cells were incubated with 0.1% hydrogen peroxide in 100% methanol (both from Wako Pure Chemicals) at 4°C for 30 min to inactivate the endogenous peroxide. After rinsing with phosphate-buffered saline (PBS), the specimens were incubated with wash buffer (2% fetal bovine serum in PBS) at 4°C for 30 min. Either monoclonal anti-human AFP antibody raised in mouse (Takara, Ohtsu, Japan) or polyclonal anti-human albumin (ALB) antibody raised in rabbit (Sigma-Aldrich) was applied to the specimens in wash buffer at 1:1000 dilution. The samples were then incubated at 4°C overnight after which, they were washed with PBS. They were then incubated with either alkaline phosphatase-linked anti-mouse antibody raised in goat (Promega, Madison, WI, USA) or alkaline phosphatase-linked anti-rabbit antibody raised in goat (Promega) in wash buffer at 1:1000 dilution, at 4°C for 2 h. After washing with PBS, the conjugates were visualized using Vector Red Substrate (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions. The specimens were then observed under a microscope (AX80) (Olympus, Tokyo, Japan).
6
Time-lapse Imaging of HMVEC Viability
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The HMVECs were grown in 8-well chamber slides (Matsunami Glass Co. Ltd., Osaka, Japan). Time-lapse images were taken every 15 min using a fluorescence microscope BZ-9000 (Keyence Co. Ltd., Osaka, Japan). Cells were cultured at 37°C in 5% CO 2 in a temperaturecontrolled humidified box mounted on the stage of an inverted microscope (MMW-001; Matsunami Glass Co. Ltd.). PI-positive cells were detected with a fluorescence filter for Texas Red.
7
Time-Lapse Imaging of HMVECs
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The HMVECs were grown in 8-well chamber slides (Matsunami Glass Co. Ltd., Osaka, Japan). Time-lapse images were taken every hour using a fluorescence microscope BZ-9000 (Keyence Co. Ltd., Osaka, Japan). Cells were cultured at 37°C in 5% CO 2 in a temperature-controlled humidified box mounted on the stage of an inverted microscope (MMW-001; Matsunami Glass Co. Ltd.). PI-positive cells were detected with a fluorescence filter for Texas Red.
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