Vero cells grown in 8-chamber slides (EMD Millipore, Burlington, MA) were infected with 10% volume of the test and inactivated ZIKV samples and incubated for 3 days post infection. Cells were fixed with ice cold acetone:methanol (1:1 v/v) for 10 min followed by 3 rinses with 1XPBS. Blocking was performed with 1% Bovine Serum Albumin (Sigma Aldrich, St. Louis, MO, USA) for 1 h at room temperature. Slides were then incubated with anti-ZIKV antibody ZV-54 (EMD Millipore, Burlington, MA, USA) diluted at 1:200 in blocking buffer for 1 h at 37 ℃. Slides were washed 3 times, 5 min per wash, with 1X PBS and incubated with AlexaFluor 488-conjugated anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) diluted at 1:500 in blocking buffer for 1 h at 37 ℃. Slides were washed with 1X PBS as mentioned above and mounted using Vectashield mounting medium containing DAPI (Vector Labs, Burlingame, CA, USA).
8 chamber slide
Manufactured by Merck Group
Sourced in Germany, Ireland
The 8-chamber slides are a laboratory equipment designed for various cell-based assays and microscopy applications. The product features eight individual chambers, allowing for the simultaneous analysis of multiple samples or experimental conditions on a single slide. The chambers are made of high-quality materials to provide a controlled environment for cell culture and observation.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Vectashield mounting medium containing dapi, by Vector Laboratories (1 mentions)
Alexa fluor 488 conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Axio observer fluorescence microscope, by Zeiss (1 mentions)
Alexa fluor plus 488, by Thermo Fisher Scientific (1 mentions)
12 well plate, by BD (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Cytiva (1 mentions)
Streptomycin, by Cytiva (1 mentions)
Plan apochromat, by Zeiss (1 mentions)
Lsm 780 confocal microscope, by Zeiss (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
F4 80, by Abcam (1 mentions)
Cd11b, by Abcam (1 mentions)
Histofix, by Carl Roth (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
9 protocols using 8 chamber slide
1
ZIKV Antibody Detection in Vero Cells
2
DNA Damage Quantification After Irradiation
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To quantify DNA damage upon irradiation, 25,000 cells were seeded on 8-chamber slides (Merck, Darmstadt, Germany). After fixation and permeabilization with 3% PFA, 0.5% Triton X-100 and 8% Sucrose in PBS, cells were blocked with 5% FBS, 5% NGS, 0.2% Triton X-100 in PBS. Next, samples were incubated with the γH2A.X antibody (1:400, Merck, 05-036) for 1 h at room temperature followed by overnight at 4 °C. After washing and incubation with the secondary antibody (anti-mouse Alexa Fluor Plus 488, 1:400, Thermo Fisher Scientific, #A32723), cells were stained with a DAPI/Hoechst33342 mix (1 µg/mL DAPI, 5 µg/mL Hoechst33342 mixed 1:1). Pictures were recorded using an Axio observer fluorescence microscope (Zeiss, kindly provided by the working group of Prof. Dr. Jendrossek, University Hospital Essen) and analyzed with the Focinator 2.0 software, which corrects for the area of the nucleus and allows for quick and semi-automated counting of γH2A.X in at least 50 cells [31 (link)].
3
Evaluating Durability and Protein Expression of PRGF Gel
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To evaluate the durability of the produced gel and to analyze the protein expression of the MNC in the PRGF, pieces of SIS (0.9 cm2) were placed in 8-chamber slides (Merck Millipore, Cork, Ireland) and 350 μL PRGF was added on top and incubated for 30–45 min in room temperature until gel was formed (Table 1 ). PRGF (1 mL) without SIS was produced as a control. The gel pieces and the PRGF-SIS constructs were transferred to 12-well plates (Becton-Dickinson) and 350 μL RPMI 1640 medium (Life Technologies) supplemented with 10% pooled inactivated human AB serum, 100 U/mL penicillin, and 100 mg/mL streptomycin (HyClone Laboratories, South Logan, UT, USA), and 20 mM L-glutamine (Invitrogen) was added per well. At days 0, 3, 7, 10, and 14, the PRGF gel and PRGF-SIS constructs were collected, washed in PBS, and transferred to 4% PFA for 3 days and thereafter paraffin embedded and sectioned. The sections were stained as above with HTX/eosin for morphology, CD34 and CD45 (both hematopoietic surface markers, 1 : 100, Dako) for surface epitope expression, Ki67 (1 : 500, Life Technologies), and for activated caspase 3 (1 : 250, Invitrogen).
4
Analyzing Microtubule Organization in MCPH1 Mutant Cells
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MCPH1 mutant and control MCF10A cells were grown overnight in 8‐chamber slides (Lab‐Tek II) coated with poly‐l ‐lysine (Sigma‐Aldrich) and fixed with 4% paraformaldehyde (PFA) for 15 min. The cells were permeabilized with 0.25% Triton X‐100 for 10 min, blocked with buffer containing 1% BSA for 30 min and labeled with anti‐tubulin beta primary antibody (clone KMX1, monoclonal, MAB3408 LOT:2452493, mouse) in +4°C overnight. Goat anti‐mouse (IgG H + L, superclonal, alexafluor 555 conjugate, A28180, Thermo Scientific) was used as the secondary antibody. Samples were co‐stained with DAPI (Ab104139 fluoroshield mounting medium with DAPI, LOT:GR201489‐2) and images were captured with Plan‐Apochromat 40×/1.4 objective with Zeiss LSM 780 confocal microscope (Carl Zeiss).
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MCPH1 mutant and control MCF10A cells were grown overnight in 8‐chamber slides (Lab‐Tek II) coated with poly‐
5
Immunostaining of Cardiac Fibroblasts and Macrophages
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BMMs and murine cardiac fibroblasts were cultivated in 8-chamber slides (Sigma-Aldrich, Germany). Cells were fixed with 4% Histofix (Roth, Germany) and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich). BMMs were stained against F4/80 (1:100, Abcam, UK) or CD11b (1:100, Abcam, UK) (Figures 1B–E Figures 1F–K
6
In Vitro Replication of Toxoplasma Tachyzoites
7
Evaluating Toxoplasma Tachyzoite Invasion
8
Immunofluorescence Cell Imaging Protocol
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For immunofluorescence cell-imaging studies, cells were plated at a density of 100,000 cells/chamber in an 8-chamber slide (Merck Millipore, Cork, Ireland) one day prior to staining in order to achieve 70% confluency. Cells were fixed in 4% freshly prepared formaldehyde for 5 min and permeabilized in PBS with %0.1 Triton X-100 for 5 min at room temperature. Cells were then incubated for 1 h in blocking solution (3% bovine serum albumin/0,08% glycine in PBS) and treated with either anti-GRP 78 (1:100, #ab21685 Abcam, Cambridge, MA, USA); anti–NF–κB p65 (1:100, #ab16502, Abcam, Cambridge, MA, USA); anti-cleaved caspase-3 (1:100, #9664, Cell Signaling Technology, Danvers, MA, USA); anti-caspase 12 (1:100, #ab62463 Abcam, Cambridge, MA, USA); overnight. The secondary antibody, Alexa Fluor-488 conjugated goat anti-rabbit (1:300, # A11008 life Technologies, USA), was applied for 1 h at room temperature and nuclei were counterstained with DAPI (Vector Laboratories Inc.,Burlingame, CA, USA) in all experiments. Slides were viewed under a fluorescence microscope (Olympus BX61 fully automated, Tokyo, Japan) and fluorescence intensity was quantified using NIH ImageJ 1.44p software.
9
RNA Isolation and Transfection for Virus Visualization
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RNA was isolated from various experimental groups using the MagMax Viral/Pathogen Nucleic Acid Isolation Kit (ThermoFisher Scientific, Waltham, MA, USA) as per the manufacturer’s protocol. Briefly, 50 µL of sample was diluted to 200 µL with sterile PBS and mixed with activated nucleic acid binding magnetic beads and proteinase K. Samples were mixed at 300 rpm for 5 min followed by incubation at 65 °C for 10 min and an additional 5 min mixing at 300 rpm. Beads were collected on the side of tube using a magnetic stand for 5 min. Supernatant was carefully removed and beads were washed with wash buffer included in the kit followed by two washes with 80% ethanol. Final elution was done in 60 µL of nuclease free water. RNA was quantified using NanoDrop (ThermoFisher Scientific, Waltham, MA, USA) and stored at −20 °C until further use. Transfection of Vero cells was performed in 8-chamber slide (EMD Millipore, Burlington, MA, USA), with equal volumes of RNA for each sample using siPORT NeoFx transfection reagent (ThermoFisher Scientific, Waltham, MA, USA). Cells were fixed 4 days post-transfection and virus infection was visualized by immunofluorescence staining as described above.
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