RAW264.7 cells were seeded in 8-chamber slides (BD Biosciences, Bedford, MA) at a density of 5×104 cells per well. Cells were incubated with or without mangiferin (25 µM) for 2 hours and then treated with TNF-α (20 ng/ml) for an additional 12 hours. Cells were fixed with a 4% paraformaldehyde solution at 20°C for 10 min. After washing in PBS, cells were permeabilized with 0.3% Triton X-100 in PBS at room temperature for 20 min. After incubation in 0.1% Triton X-100 blocking buffer, 1% BSA, and 3% donkey serum, cells were incubated with a rabbit NF-κB p65 antibody (1:50) at 4°C overnight and then incubated with an Alexa Fluor 488-conjugated anti-rabbit IgG secondary antibody (1:500) at room temperature for 45 min. DAPI at a concentration of 1 µg/ml in PBS was added to stain the nuclei. Fluorescence photographs were obtained using a CKX41 inverted fluorescence microscope (Olympus, Shanghai, China).
8 chamber slide
Manufactured by BD
Sourced in United States
The 8-chamber slides are laboratory equipment used for cell culture and analysis. They provide a multi-well format with eight separate chambers for conducting parallel experiments or observations. The slides are typically made of glass or plastic and are designed for use with microscopes and other laboratory instruments.
Automatically generated - may contain errors
Lab products found in correlation
Axio vert a1, by Zeiss (2 mentions)
Anti human transferrin receptor antibody, by Thermo Fisher Scientific (2 mentions)
Rodamine red, by Jackson ImmunoResearch (2 mentions)
Alexa fluor 555 conjugated, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
Ckx41 inverted fluorescence microscope, by Olympus (1 mentions)
Pe rat anti human cd184, by BD (1 mentions)
Axioimager apotome, by Zeiss (1 mentions)
Envision dual link system hrp, by Agilent Technologies (1 mentions)
3 3 diaminobenzidine dab tablets, by Merck Group (1 mentions)
Goat serum, by Merck Group (1 mentions)
Eclipse te2000 u, by Nikon (1 mentions)
Alexa fluor 546 phalloidin, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by ProSciTech (1 mentions)
Streptavidin cy3, by Merck Group (1 mentions)
Saponin, by Merck Group (1 mentions)
Biotinylated donkey anti sheep antibody, by Jackson ImmunoResearch (1 mentions)
Fc receptor blocking antibody, by BD (1 mentions)
15 protocols using 8 chamber slide
1
NF-κB Activation in RAW264.7 Cells
2
Immunostaining of CD184 in ADHLSCs
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ADHLSCs were plated at passage 4 on 8-chamber slides (BD Biosciences) at a density of 5,000 cell/cm2. Upon reaching 70% confluence, they were fixed with 4% paraformaldehyde for 15 minutes. After 2 washes with DPBS, ADHLSCs were blocked with DPBS-BSA 5% for 1 hour. Some of the samples were permeabilized with Triton 0.1% DPBS-BSA 1.5% buffer (BD Biosciences) for 20 minutes at 4°C. The cells were then stained with a PE rat anti-human CD184 for 2 hours at 4°C (BD Biosciences) and rinsed 3 times with DPBS. Finally, samples were embedded in ProLong Gold with DAPI (BD biosciences). Pictures were taken with an Axio Imager + ApoTome (Zeiss) at a 20x objective and analyzed with AxioVision software.
3
PD-L1 Immunocytochemical Detection Protocol
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For immunocytochemical DAB staining of PD-L1, cells were seeded in 8-chamber slides (BD Biosciences). Cells were fixed in 2% paraformaldehyde (PFA) (minimum 37%) (Merck) for 10 min. Prior to staining, endogenous peroxidase activity was blocked by incubating the cells with 10% H2O2 (hydrogen peroxide solution) (Sigma–Aldrich) and 10% MeOH (Carl Roth) for 10 min at room temperature. Cells were incubated for 1 h with 5% goat serum (Sigma–Aldrich) in PBS (Life Technologies, Carlsbad, CA, USA) as a blocking solution to prevent nonspecific antibody binding. The primary anti-PD-L1 antibody (rabbit mAb, clone E1L3N, #13684, CST) was diluted to 3 µg/mL in 3% BSA in PBS (Life Technologies) and incubated overnight at 4 °C. A rabbit monoclonal IgG antibody (#3900, CST) was used as an isotype control at the same concentration. For signal detection, slides were incubated for 10 min with EnVision™+ Dual Link System-HRP (DAKO, Agilent, Santa Clara, CA, USA). Visualization of immunoreactivity was performed using 3,3’-diaminobenzidine (DAB) tablets (Sigma–Aldrich). The samples were incubated for 30 min. The samples were then dehydrated by an ascending alcohol series and coverslipped with Entellan. Samples were digitally photographed in bright field illumination at 20-fold magnification under a microscope (Nikon Eclipse TE2000-U, Nikon, Minato, J).
4
Immunofluorescent Staining of PD-L1 in Cells
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IF staining was performed in 8‐chamber slides (BD Biosciences). Cells were washed thoroughly with PBS (DPBS, including calcium and magnesium, Gibco) prior to fixation with 4% paraformaldehyde (PFA) for 10 min at RT. Before antibody incubation, cells were washed thoroughly again, and nonspecific binding sites were covered with a blocking solution containing 3% BSA (Biomol) and 0.1% Triton X‐100 (Sigma‐Aldrich). Primary antibody incubation was performed with anti‐PD‐L1 (rabbit mAb, clone E1L3N, #13684, CST) in antibody diluent containing 1% BSA (Biomol) and 0.1% Triton X‐100 (Sigma‐Aldrich) overnight at 4 °C. For signal detection of the target protein host‐specific F(ab′)2‐Goat anti‐Rabbit IgG (H + L), cross‐adsorbed secondary antibody (Thermo Scientific) was incubated for 1 h at 37 °C. Cytoskeleton was stained with Alexa Fluor™ 546 Phalloidin (Thermo Scientific) for 20 min at 37 °C. Slides were mounted with VECTASHIELD Antifade Mounting Medium with DAPI (Biozol Diagnostica Vertrieb GmbH, Eching, Bayern) and covered with a high precision microscope cover glass (Marienfeld Superior, Lauda‐Königshofen, Germany).
5
Immunofluorescence Staining of Cells
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Cells were seeded at 15,000 to 30,000 cells per well in duplicate in 8-chamber slides (BD Biosciences, San Jose, CA, USA). The next day, cells were washed twice with 1X PBS and fixed in 4% paraformaldehyde for 15 min. Following 2 washing step with 1X PBS, cells were permeabilised for 15 min using 0.2% Triton-X100, then washed again and incubated for 30min in 3% bovine serum albumin (BSA) in TBS-Tween. Cells were incubated at 4 _ C overnight under constant agitation in antibodies of choice (diluted in 3% BSA in TBS-Tween) or 3% BSA in TBS-Tween only as negative control.
Afterwards, cells were washed twice using 1X PBS and then incubated with secondary fluorescently tagged antibody for 90min at 37C º protected from light. Following several washing steps in 1X PBS, the chambers were removed from the slides, and cells were mounted in Vectashield mounting medium containing 4,6-diamidin-2-phenylindole (DAPI; Vector Laboratories; Burlingame, CA, USA). Images were acquired in Nikon Centre (King's College London) at 60X or 100X magnifications. Antibodies used for immunofluorescence were EGFR, 53BP1 and gammaH2AX (Cell signaling CST).
Afterwards, cells were washed twice using 1X PBS and then incubated with secondary fluorescently tagged antibody for 90min at 37C º protected from light. Following several washing steps in 1X PBS, the chambers were removed from the slides, and cells were mounted in Vectashield mounting medium containing 4,6-diamidin-2-phenylindole (DAPI; Vector Laboratories; Burlingame, CA, USA). Images were acquired in Nikon Centre (King's College London) at 60X or 100X magnifications. Antibodies used for immunofluorescence were EGFR, 53BP1 and gammaH2AX (Cell signaling CST).
6
Visualizing CLIC1 and RhoA in BMDCs
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Resting BMDCs or BMDCs 5 min after initiation of synchronised phagocytosis were fixed with 4% paraformaldehyde (Cat #C004, ProSciTech) on 8-chamber slides (Cat #354108, BD Biosciences), then permeabilised with 0.05% saponin (Cat #S4521, Sigma-Aldrich) (Jiang et al., 2012 (link)). After blocking with 2% IgG free BSA (Cat #001-000-161, Jackson ImmunoResearch Labs) and 1 µg/ml Fc receptor blocking antibody (Cat #553142, BD Biosciences), the cells were stained for; CLIC1 and RhoA. Briefly, BMDCs were firstly stained with 1:100 rabbit anti-mouse RhoA antibody then 1:100 donkey anti-rabbit Cy2 antibody (Cat#ab6940, Abcam) followed by an in-house-derived 1:1000 sheep anti-mouse CLIC1 (Jiang et al., 2012 (link)), then 1:100 biotinylated donkey anti-sheep antibody (Cat #713-065-003, Jackson ImmunoResearch Labs) and finally streptavidin Cy3 (Cat#S6402, Sigma-Aldrich) (Jiang et al., 2012 (link)). Confocal images were obtained on a Leica TCS SP confocal microscope (Leica Microsystems, Germany) and processed using ImageJ64 (NIH, imagej.nih.gov/ij/download/).
7
Immunocytochemistry of BRD4 in Cells
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50,000 cells were seeded into 8-chamber slides (BD Biosciences). On the next day, cells were washed twice with 1× PBS and fixed with 4% paraformaldehyde solution for 20 minutes. Cells were then washed with 1× PBS thrice and were subjected to permeabilisation using 0.2% Triton-X100, then washed three times and incubated with 1% BSA in PBS for 60 minutes. Cells were incubated overnight at 4 °C with antibody of choice diluted in 1× PBS or simply 1× PBS as a negative control. Afterwards, cells were washed three times with 1× PBS and incubated with fluorescence-tagged secondary antibody for 90 minutes at room temperature protected from light. Following three times washing with 1× PBS, the chamber was removed from the slide and mounted with Vectashield mounting medium containing 4,6-diamidin-2-phenylindole (Vector Laboratories). Images were acquired using Zeiss LSM 700 microscope. The dilution used for BRD4 antibody was 1:200. Fluorescence intensities were measured using ImageJ 1.53k.
8
Seeding Primary Human Hepatocytes
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Sterilized μHEP slides or plates, or commercially available culture devices such as 8-chamber slides (BD, Franklin Lakes, NJ), were unpackaged in a sterile field and placed in a Petri dish to serve as a lid and control evaporation. The day prior to PHH seeding, wells were collagen coated with 40 μL of 15 μg μL -1 rat tail collagen I (BD) in sterile filtered 0.02 N acetic acid (Thermo Fisher Scientific, Waltham, MA), then left overnight at 37 °C. Immediately prior to seeding the collagen solution was washed out thrice with sterile PBS and then filled with 20 μL In Vitro GRO® CP plate media (BioIVT, Westbury, NY) supplemented with 1× Pen-Strep-Neo solution (Thermo Fisher Scientific) and 20 μM gentamicin (Thermo Fisher Scientific). Vials of cryopreserved PHHs (BioIVT or Yecuris, Tualatin, OR) were thawed by immersion in a 37 °C water bath for 2 minutes, sterilized by 70% ethanol in a sterile field, and contents added directly to 4 mL plate media. Live and dead cells were quantified by trypan blue exclusion on a Neubauer improved hemocytometer, the PHH density was set to 1 × 10 3 live cells per μL, and 18 μL cell suspension was added to each well. Media was exchanged thrice weekly with CP media with antibiotics.
9
Immunofluorescence Analysis of EGFR and FPR2
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30,000 cells were seeded in duplicate in 8-chamber slides (BD Biosciences, San Jose, CA, USA). The next day, cells were washed twice with 1Â PBS and fixed in 4% paraformaldehyde for 15 min. Following 2 washing step with 1Â PBS, cells were permeabilised for 15 min using 0.2% Triton-X100, then washed again and incubated for 30 min in 3% bovine serum albumin (BSA) in TBS-Tween. Cells were incubated at 4 C overnight under constant agitation in antibodies of choice (diluted in 3% BSA in TBS-Tween) or 3% BSA in TBS-Tween only as negative control. Afterwards, cells were washed twice using 1Â PBS and then incubated with secondary fluorescently tagged antibody for 90 min at 37 C protected from light. Following several washing steps in 1Â PBS, the chambers were removed from the slides, and cells were mounted in Vectashield mounting medium containing 4 0 ,6-diamidin-2-phenylindole (DAPI; Vector Laboratories; Burlingame, CA, USA). Images were acquired on an Olympus BX61 at 60Â magnification. Antibodies used for immunofluorescence were EGFR F4 and FPR2. The FPR2 antibody was kindly provided by Prof Mauro Perretti, William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London, UK.
10
LPS-Induced NF-κB Activation in BV-2 Cells
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The BV-2 cells were seeded in 8 chamber slides (BD Biosciences, San Diego, CA, USA) at a density of 2x10 5 cells/ml. Tweety-four hours later, the cells were treated with or without ISO I (100 µM) for 2 h followed by stimulation with LPS (200 ng/ml) for 20 h. Thereafter, they were washed with phosphate-buffered saline (PBS), fixed with 4% PFA, permeabilized and blocked in PBS with 5% normal donkey serum and 0.1% Triton X-100. Half an hour later, cells were incubated with p-NF-κB p65 antibody overnight at 4̊C. After washing in PBS, they were further incubated with Alexa-594 conjugated secondary antibody at room temperature for 1 h. At last, cells were sealed in medium with DAPI. Fluorescence images of the cells were acquired using an Olympus DX81 fluorescent microscope (Olympus, Tokyo, Japan).
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