Primary FVB/n keratinocytes were plated on 8-well μ-slides (IBIDI) and transduced with v-RasHa for 2 days, followed by 1 ng/ml TGFβ1 for 48h. To measure ER content and expansion, the cells were stained with 1 μM ER-Tracker™ Green (Invitrogen) in sterile HBSS for 30 minutes at 37°C. Cells were mounted with DAPI mounting media (VectorLabs) and were visualized with LSM880 Fluorescence microscope with Airyscan (Zeiss) using the 63X oil-immersion objective. Z-stacking was performed by ImageJ. Absolute ER Area and background- and area- corrected mean fluorescence intensity were quantitated from at least 30 to 50 cells per condition using ImageJ. To measure accumulation of unfolded proteins, the keratinocytes were stained with freshly prepared 5 mM Thioflavin T (Sigma) solution as described previously51 (link). The cells were mounted with DAPI mounting media (VectorLabs) and visualized with Keyence BZ-9000 fluorescence microscope using 40X oil-immersion objective. Z-stacks were generated using ImageJ and background- and area- corrected mean fluorescence intensity was calculated from at least 30 to 50 cells per treatment condition. Statistical outliers determined by the IQR approach were removed from analysis.
Dapi mounting media
Manufactured by Vector Laboratories
Sourced in United States, United Kingdom
DAPI mounting media is a fluorescent stain used for DNA detection and visualization in biological samples. It binds strongly to the minor groove of double-stranded DNA, emitting blue fluorescence when excited by ultraviolet light. The mounting media serves to preserve the fluorescent signal and protect the sample.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield hardset, by Vector Laboratories (2 mentions)
Tom20 sc 11415, by Santa Cruz Biotechnology (2 mentions)
Anti ha antibodies 2367 and 3724, by Cell Signaling Technology (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Primary anti 53bp1 antibody, by Santa Cruz Biotechnology (2 mentions)
Alexa 488 labeled secondary antibody, by Thermo Fisher Scientific (2 mentions)
Pna cy3 telo c probes, by Agilent Technologies (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Spautin 1, by Merck Group (1 mentions)
Alexa fluor 488 labeled goat anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
Eclipse e800, by Nikon (1 mentions)
Mitotracker green, by Thermo Fisher Scientific (1 mentions)
Bodipy, by Thermo Fisher Scientific (1 mentions)
Bx60 fluorescence microscope, by Olympus (1 mentions)
Dfc360 fx, by Leica (1 mentions)
Dm5500b, by Leica (1 mentions)
Vectashield plus antifade mounting medium, by Vector Laboratories (1 mentions)
31 protocols using dapi mounting media
1
Quantifying ER Expansion and Unfolded Protein Accumulation
2
BKPyV Infection and Autophagy Modulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
5×103 cell were plated in 96-well culture plates. The following day, cells were treated with multiple drugs that influence autophagy levels, rapamycin, 3-methyladenine, bafilomycin-A, DMSO, and spautin-1 (Sigma) before and after being infected with BKPyV (MOI:5) for 1 h at 37 °C. 72 h post infection, cells were fixed using 2% paraformaldehyde (EMB) and permeabilized using 0.5% Triton X-100 (Shelton Scientific). To detect the number of infected cells expressing viral protein, cells were incubated with the anti-V antigen (V-Ag) monoclonal antibody PAB597, secondary Alexa-Fluor-488-labeled goat anti-mouse antibody (Molecular Probes), and then counterstained with DAPI-mounting media (Vector Labs). The PAB597 hybridoma produced a monoclonal antibody against the SV40 major capsid protein VP1 that cross-reacts with BKPyV VP1. Infected cells were scored using a Nikon epifluorescence microscope (Eclipse E800; Nikon, Inc.). Approximately 20,000 cells, the entire surface of the 96 well, were screened for V-Ag expression.
3
Immunostaining of Murine Small Intestine
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Small intestines were isolated from mice shortly after euthanization and flushed with PBS, the gut was then inverted onto a wooden skewer and placed in 10% neutral buffered formalin (NBF, Merck) solution for 4 h. The tissue was sliced longitudinally to remove from the skewer and tissue was then rolled up beginning at the ileum using the Swiss roll method as previously published76 . The tissue was stored in NBF solution O/N then placed into 70% ethanol prior to paraffin embedding. Thin sections of paraffin-embedded tissue were de-waxed and stained with anti-DCAMLK1 at 1:1000 (Abcam, UK) and secondary stained using anti-rabbit-FITC, control slides were incubated with rabbit IgG (Abcam, UK). Once stained, slides were mounted in DAPI mounting media (Vector Laboratories, UK) and imaged using the 10x objective on a Leica DiM8 microscope (Leica, Germany); images were analysed using ImageJ/Fiji.
4
Mitochondrial Morphology and Function Assessment
5
FITC-Labeled Peptide Uptake Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
FITC-labeled peptides were dissolved in water and, if necessary, acetic acid was used to help dissolve the peptide (final acetic acid concentration in experiment <0.2% v/v.) Final peptide concentrations were determined by spectrophotometric analysis at 490 nm (pH 7.5; ∼20 times diluted in 1 M Tris-HCl buffer [pH 7.5]).
Human control myotubes and immortalized human cardiomyocytes were washed twice with PBS and incubated with 2.25 μM FITC-labeled peptides in serum-free media for 3 hr at 37°C and 5% CO2. Cells were washed three times with PBS and fixed with cold methanol (−20°C) for 5 min (human control myotubes) or 10 min (human cardiomyocytes). Subsequently, the glass slides were shortly air-dried and embedded on microscope slides with Vectashield hard set containing DAPI mounting media (Vector Laboratories, UK). After drying for 30 min, slides were analyzed with fluorescence microscopy, 20× magnification (Leica DM5500 B) using a CCD camera (Leica DFC 360 FX).
Human control myotubes and immortalized human cardiomyocytes were washed twice with PBS and incubated with 2.25 μM FITC-labeled peptides in serum-free media for 3 hr at 37°C and 5% CO2. Cells were washed three times with PBS and fixed with cold methanol (−20°C) for 5 min (human control myotubes) or 10 min (human cardiomyocytes). Subsequently, the glass slides were shortly air-dried and embedded on microscope slides with Vectashield hard set containing DAPI mounting media (Vector Laboratories, UK). After drying for 30 min, slides were analyzed with fluorescence microscopy, 20× magnification (Leica DM5500 B) using a CCD camera (Leica DFC 360 FX).
6
Immunofluorescence Imaging of Transfected Cells
7
Immunofluorescence Imaging of Transfected Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
COS-7 or HeLa cells were seeded to 75% confluence onto glass coverslips and transfected using Lipofectamine 2000 (Invitrogen) according to manufacturer’s protocol. Cells were fixed with paraformaldehyde for 20 min, permeabilized with cold methanol for 5 min and stained with anti-myc (#2276 and #2278) or anti-HA antibodies (2367 and 3724) from Cell Signaling.Tom20 (SC-11415) from Santa Cruz was used to label mitochondrial outer membrane. Corresponding Alexa-fluor-conjugated secondary antibodies (Life Technologies) were used for detection. Coverslips were mounted onto slides with DAPI mounting media (Vectashield), and imaged with a confocal microscope, Zeiss LSM710, 63X/1.4 oil objective with a pinohole of less than one Airy unit and processed with Fiji (Image J).
8
Retinal Immunofluorescence Imaging Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole eyes were excised, and corneas were punctured, followed by incubation in 4% paraformaldehyde (PFA, pH 7.5) for 30 min. Eyes were washed with PBS and incubated at 4 °C in 30% sucrose solution containing 0.05% sodium azide. Eyes were embedded in optimal cutting temperature compound, flash frozen, and sectioned. Cryosections (10 μm) were fixed in 2% PFA, permeabilized in PBS with 0.1% Triton-X-100. To visualize NF-κB nuclear localization, MIO-M1 cells were cultured on chamber slides (CELLTREAT) for 24 h prior to exposure to hyperglycemia conditions. Cells were then fixed in 4% PFA and permeabilized with PBS with 0.1% Triton-X-100. Sections or cell monolayers were then blocked with 10% normal donkey serum and labeled with the appropriate antibodies (Table S1 ). Slides were mounted with Vectashield Plus Antifade Mounting Medium with DAPI mounting media (Vector labs). Images were captured using an SP8 confocal laser microscope (Leica Microsystems) with frame-stack sequential scanning. ImageJ (National Institutes of Health) was used to estimate macrophage counts from three separates fields of view per retina. Thresholds were set to isolate CD80- or F4/80-positive cells, and particles were counted. Single-stained population counts were also confirmed manually.
9
Immunofluorescence Staining of CDH1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were cultured on untreated coverslips in 6-well plates (2 × 104 cells/well) and treated as indicated. Coverslips were fixed for 30 min in methanol at −20 °C, washed once with acetone, and then 5 times with PBS. Samples were blocked and permeabilized for 30 min in blocking buffer (5% normal goat serum, 0.3% TritonX-100 in PBS). Cells were incubated with anti-CDH1 antibody (BD Biosciences, 610181; diluted 1:100 in blocking buffer) for 1 h at RT. Secondary antibodies (BD Biosciences, 1:500) were incubated for 1 h at RT. Coverslips were mounted in DAPI mounting media (Vector Labs). Images were obtained using the Zeiss Axiovert 200 microscope with a 10x objective and the AxioVision software version 4.6.3 SP1.
10
Assessing Macrophage Infiltration in Ischemic Muscle
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!