The largest database of trusted experimental protocols

92 protocols using dapi mounting medium

1

Chromosome Enumeration Probes for FISH

Check if the same lab product or an alternative is used in the 5 most similar protocols
Chromosome enumeration probes (CEP) 8 (Vysis, Des Plaines, IL, USA) was denatured in hybridization buffer (0.15 M sodium chloride, 0.015 M sodium citrate, 70% formamide) at 73°C for 5min. The probe was then incubated with a slide in a humidified chamber at 42°C overnight followed by washing with 0.3% (w/v) NP-40 in 0.4x saline-sodium citrate (SSC; 1x SSC containing 150 mM NaCl and 15 mM Na3Citrate) at 73°C for 2 min and with 2x SSC/0.1% NP-40 at room temperature for 30 s. After briefly drying, slides were analyzed after mounting with DAPI mounting medium (Vector Labs) under fluorescence microscope.
+ Open protocol
+ Expand
2

Immunofluorescence and Flow Cytometry Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cells were fixed in 4% paraformaldehyde-PBS solution. For visualization of intracellular markers, cells were permeabilized with 0.1% Triton X-100-PBS solution, blocked with 2% BSA-PBS solution for 1 hr at room temperature, and incubated overnight at 4°C with primary antibodies as listed in Table S4. Appropriate fluorescence-tagged secondary antibodies (Molecular Probes and Vector Laboratories) were used for visualization. Cells were mounted in DAPI mounting medium (Vector Laboratories), and images were obtained using an Olympus IX71 microscope equipped with a DP71 digital camera or Olympus FSX100 system. MetaMorph Microscopy Automation and Image Analysis Software (Molecular Devices) were used to count positive cells in seven to ten randomly selected images at a final magnification of ×200 from each of three independent experiments. For flow cytometry, cells were dissociated into single cells using GIBCO Cell Dissociation Buffer (Invitrogen) and incubated in 1% BSA-PBS solution with the appropriate antibodies as listed in Table S4. For unconjugated primary antibodies, the cells were incubated with Alexa-Flour 488-conjugated anti-mouse IgG/IgM (Molecular Probes) for raising suitable secondary antibodies. Flow cytometry was performed using FACSCalibur (BD Biosciences) and analyzed using WinMDI 2.8 or FACSVerse (BD Biosciences) and FlowJo software.
+ Open protocol
+ Expand
3

Immunohistochemistry of Liver CD36 Expression

Check if the same lab product or an alternative is used in the 5 most similar protocols
Liver sample preparation and sectioning was performed similar to the procedures described for Oil Red O staining above, except 16 μm sections were used. Liver sections were dried at room temperature and then equilibrated in PBS solution for 20 min. Sections were blocked with 0.3% BSA solution in PBS and incubated with anti-CD36 (1:200, Novus Biologicals, Littleton, CO) overnight. They were then washed three times with PBS for 10 min and subsequently incubated with a goat anti-rabbit antibody (1:1000, Abcam, Cambridge, MA), which was followed by 3 consecutive 10 min PBS washes. Sections were then dried at room temperature and counterstained with DAPI mounting medium (Vector Laboratories, Burlingame, CA). Images were obtained using a light microscopy (Olympus BX43, Center Valley, PA).
+ Open protocol
+ Expand
4

Phagocytosis Assay in Murine Macrophages

Check if the same lab product or an alternative is used in the 5 most similar protocols
Macrophages derived from ACE10 mice were seeded into each chamber of 8-chamber culture slides (BD bioscience) in culture medium. Phagocytosis was performed by adding 1μm latex beads (Sigma,1:500 dilution) into the cells and co-incubating for 1 hour at 37 °C. After extensive washing with PBS, cells were fixed with 4% PFA/PBS for 10 min followed by blocking and permeabilization with 1% BSA and 0.1% Triton X-100 in PBS for 1 hour at room temperature (RT). Then, primary antibodies were used for staining at RT for 2 hours followed by treating the cells with secondary antibodies for another 2 hours at RT. After PBS washing, chamber walls were removed and coverslips were mounted with DAPI- mounting medium (Vector). Images were acquired using a laser scanning confocal microscope (Olympus FV10i). A secondary anti-rabbit antibody used to stain rabbit-anti-ACE IgG was Alexa fluor 488-conjugated (Abcam), while other secondary antibodies were all anti-mouse IgG conjugated with Alexa fluor 594 (Abcam).
+ Open protocol
+ Expand
5

Multicolor Immunofluorescence Staining of FFPE Tissue

Check if the same lab product or an alternative is used in the 5 most similar protocols
4 μm paraformaldehyde-fixed paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated followed by antigen retrieval in 10 mM sodium citrate and a blocking step with 10% normal donkey serum/5% BSA/PBS. Chicken anti-GFP (Abcam), rabbit anti-Salmonella O4 antigen (Abcam), rat anti-LAMP1 (Developmental Studies Hybridoma Bank), rabbit anti-cleaved caspase-3 (Cell Signaling), rabbit anti-cleaved caspase-8 (Cell Signaling), rat anti-PMN (Ly6-6B2, SeroTec) and mouse anti-E-cadherin (BD Transduction Laboratories) antibodies as well as the indicated fluorophore-conjugated secondary antibodies (Jackson ImmunoResearch) were used. Fluorescein-conjugated (Vector) or AF647-conjugated (Invitrogen) Wheat Germ Agglutinin (WGA) was used to detect the mucus layer. Slides were subsequently mounted in DAPI mounting medium (Vector) and images were taken using a Zeiss ApoTome.2 system microscope connected to a Axiocam 506 digital camera. Images were formatted using the ZEN 2.3 imaging software.
+ Open protocol
+ Expand
6

Immunostaining of Osteogenic Markers

Check if the same lab product or an alternative is used in the 5 most similar protocols
Slides were permeabilized with 0.5% Triton X-100. After antigen
retrieval, blocking was carried out with 5% goat serum (S-1000, Vector) for 1h
at room temperature. Slides were incubated with primary antibodies at 4°C
overnight. Primary antibodies included anti-Osterix (ab22552, Abcam), anti-Runx2
(ab23981, Abcam) and anti-Cathepsin K (ab19027, Abcam). Following PBS washing,
slides were incubated with Cyanine5 conjugated goat anti-rabbit secondary
antibody (A10523, Invitrogen) for 1h at room temperature, then mounted with DAPI
mounting medium (Vector Laboratories).
+ Open protocol
+ Expand
7

Immunohistochemical Analysis of Melanoma Markers

Check if the same lab product or an alternative is used in the 5 most similar protocols
De-paraffinized, ethanol-rehydrated tissue sections were stained with monoclonal antibodies (mAb) against surface markers of melanoma (HMB45; Pierce™, Cat.#MA1-34759; dilution 1:50), IL-6 (BioXCell, Inc. Cat.#BE0046; dilution 1:100), OPN (R&D system, Inc.Cat.#AF808; dilution 1:500), and Ki-67 (Pierce™, Cat.#PA5-19462; dilution 1:50). Pre-treatment and revelation procedure were performed following the instruction of VECTASTAIN ABC Kit (Vector Laboratories, Inc. Cat.#PK-4001, 4002, and 4005). For fluorescence immunostaining, slides were treated with 0.2% Triton X-100 and blocked with 5% normal serum control after antigen retrieval with citrate buffer (10mM Citric Acid, 0.05% Tween 20, pH 6.0), and incubated with the primary antibody and then secondary antibodies. Slides were covered using a DAPI mounting medium (Vector Laboratories Inc. Cat.#H-1200). Images were acquired using a Nikon Eclipse 80i microscope, equipped with Sony DXC-390P digital camera and NIS-Elements BR2.2 software.
+ Open protocol
+ Expand
8

Oil Red-O Staining of Frozen Tumor Sections

Check if the same lab product or an alternative is used in the 5 most similar protocols
10μm frozen tumor sections were fixed with 4% PFA in PBS for 10 minutes at room temperature. Slides were washed twice with 1X PBS and permeabilized with 1X PBST for 5 minutes at room temperature with gentle rocking. Slides were washed three times with 1X PBS for ten minutes each at room temperature, then stained with Oil Red-O solution (4 g/l ORO powder in 60% isopropanol) at room temperature for 10 minutes. Slides were washed twice with 1X PBS and counterstained with Vectashield with DAPI mounting medium (Vector Labs).
+ Open protocol
+ Expand
9

Immunofluorescence Staining of DNA Damage

Check if the same lab product or an alternative is used in the 5 most similar protocols
Immunofluorescence staining was performed by fixing cells with prechilled (–20°C) acetone-methanol for 15 minutes, followed by addition of primary antibodies to anti-γH2AX (Cell Signaling, 1:100) or anti-histone H3 S10 phosphorylation (Upstate, 1:250) at 4°C overnight. After rinsing, FITC-Donkey anti-rabbit IgG (1:200, Jackson Immuno Research Lab) was applied for 1 hour at room temperature. Slides were subsequently covered with the DAPI mounting medium (VECTOR Lab Inc.). Images were then acquired with a fluorescent microscope (Carl Zeiss, Axiovert 200).
+ Open protocol
+ Expand
10

Vinculin Localization in Cell Cultures

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cells were grown in 8-well chamber slides (Thermo Scientific, Waltham,
MA) for 24 hours before fixation and antibody staining, as previously
described26 (link). Briefly,
cells were incubated with a 1:400 dilution of primary mouse anti-vinculin
antibody (Sigma, St. Louis, MO) for 1 hour before addition of anti-mouse
AlexFlour-488 conjugated secondary antibody. Cells were fixed with rhodamine
phalloidin, according to the manufacturer’s protocol (Thermo Scientific,
Waltham, MA) before mounting using DAPI mounting medium (Vector Laboratories,
Burlingame, CA). Images were taken using a Zeiss LSM 710 confocal
microscope.
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!