Sterilized coverslips were pre‐coated with 0.01% Poly‐l ‐Lysine (P4832, Sigma) for 5 min and then washed with PBS and dried for 2 h. 1 × 105 HCT116 R248W cells were added and incubated for 72 h. Cells were fixed for 15 min with Pierce™ Methanol‐free 16% Formaldehyde (Thermo Fisher) diluted to 4%, washed with PBS, permeabilized 2 min with 0.2% Triton X, washed, and blocked for 60 min. Blocking buffer contained 2% Bovine Serum Albumin (BSA), 5% glycerol, and 0.2% Tween‐20 in PBS. Primary antibody D7O8N against MRP1 diluted 1:200 (14685S, Cell Signaling, USA) was prepared in blocking buffer and incubated at 4°C over night. Coverslips were washed with PBS, anti‐rabbit (A‐110008, Thermo Fisher) and anti‐rat (A‐110006, Thermo Fisher) Alexa Flour® 488 secondary antibodies were diluted 1:500 in blocking buffer together with Phalloidin‐Atto 647N 1:500 (65906‐10NMOL, Sigma) and incubated for 1 h. Coverslips were washed and mounted with VECTASHIELD HardSet Antifade Mounting Medium with DAPI (H‐1500 Vector Laboratories). Next day samples were imaged by Zeiss AxioObserver Z1‐inverted microscope equipped with Axiocam 506 mono camera using the 63× oil immersion lens and processed using ZEN software by Zeiss.
Vectashield hardset antifade mounting medium with dapi
Manufactured by Vector Laboratories
Sourced in United States, Canada, United Kingdom
VECTASHIELD HardSet Antifade Mounting Medium with DAPI is a reagent used for mounting fluorescently labeled samples onto microscope slides. It is designed to inhibit photobleaching and maintain the intensity of fluorescent signals. The medium contains DAPI, a fluorescent DNA-binding dye, which allows for the visualization of cell nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Apotome, by Zeiss (6 mentions)
Lsm 700, by Zeiss (5 mentions)
Donkey serum, by Jackson ImmunoResearch (4 mentions)
Lsm 710 confocal, by Zeiss (4 mentions)
Bz x800 microscope, by Keyence (4 mentions)
Confocal microscope, by Leica camera (3 mentions)
Cy3 conjugated, by Jackson ImmunoResearch (3 mentions)
Fitc conjugated, by Jackson ImmunoResearch (3 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Fibronectin coated 8 chamber glass slides, by Thermo Fisher Scientific (2 mentions)
Rabbit αflag antibody, by Merck Group (2 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg antibody, by Jackson ImmunoResearch (2 mentions)
Pser129 α syn, by BioLegend (2 mentions)
18 mm round glass coverslips, by Avantor (2 mentions)
Bz x viewer, by Keyence (2 mentions)
Bz x analyzer imaging system, by Keyence (2 mentions)
101 protocols using vectashield hardset antifade mounting medium with dapi
1
Immunofluorescence Imaging of MRP1 in HCT116 Cells
2
Visualizing FKBP13 and KDEL in Tunicamycin-Treated Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
J558 cells were treated with 10 μg/ml tunicamycin (Tm) or DMSO for 48 h. The cells were plated on coverslips coated with poly-l -lysine (Sigma-Aldrich) for 1 h, fixed with 4% paraformaldehyde in PBS for 15 min, and permeabilized with 0.5% Triton X-100 in PBS for 15 min. After blocking with 2% bovine serum albumin in PBS for 1 h, the cells were incubated with a mixture of goat anti-FKBP13 (Santa Cruz) and mouse anti-KDEL antibodies (Abcam) for 2 h and then with a mixture of anti-goat IgG conjugated with Alexa Fluor 555 and anti-mouse IgG conjugated with Alexa Fluor 488 (Thermo Fisher Scientific). Cells were washed with PBS three times between steps. The coverslips were mounted on slides using VECTASHIELD Hardset Antifade Mounting Medium with DAPI (Vectorlabs). Fluorescence images were visualized using a confocal microscope (Zeiss LSM 700) and analyzed using ZEN 2012 SP2 (blue edition, ver.1.1.2.0).
3
Visualization of Siglec F-Positive Eosinophils
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen sections were dried at room temperature and fixed in cold 4% paraformaldehyde (Sigma) in dH2O at −20° for 5 min. Slides were rehydrated in wash buffer, 0·05% bovine serum albumin (BSA) (Sigma) in PBS, for 5 min. Slides were blocked with 5% BSA in PBS, washed in PBS and avidin/biotin activity was blocked according to manufacturer's instructions with the Avidin/Biotin Blocking System (BioLegend, London, UK). Slides were stained and co‐stained with biotinylated anti‐Siglec F antibody (Bio‐Techne, Abingdon, UK) and fluorescein isothiocyanate‐conjugated cytokeratin antibody (Sigma). Sections were then stained with Streptavidin‐horseradish peroxidase solution (Vector Laboratories, Peterborough, UK), washed with PBS and stained with tyramide‐Cy3 (Perkin Elmer, Seer Green, UK) before being left to air dry. Slides were mounted with VECTASHIELD® HardSet Antifade Mounting Medium with DAPI (Vector Laboratories) and imaged with a Nikon ECLIPSE Ci‐L microscope with a DS‐Fi3 Microscope Camera (Nikon, Kingston upon Thames, UK). Red cells were positive for Siglec F (eosinophils) and green cells were positive for cytokeratin (epithelial cells). Co‐localized staining (i.e. yellow cells) were discarded as tuft cells.
4
Quantification of Rickettsia Attachment and Invasion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed using 4% paraformaldehyde (PFA) for 20 minutes and washed thoroughly with PBS. To distinguish between cell attachment and invasion, extracellular bacteria were stained with rabbit anti-Rickettsia I1789 antibody (provided by Ted Hackstadt) followed by the secondary antibody, Alexa 594 goat anti-rabbit (Invitrogen, A11005; 1:1000 dilution). To subsequently stain all bacteria (intracellular and extracellular), cells were permeabilized and again stained with rabbit anti-Rickettsia I1789 antibody followed by the secondary antibody, Alexa 488 goat anti-rabbit (Invitrogen, A11008; 1:1000 dilution). Coverslips were mounted with VectaShield HardSet antifade mounting medium with DAPI (Vector Laboratories Inc.) for nuclear staining. Samples were visualized and quantified using a Nikon A1 microscope (S10RR027535).
For growth curves, cells were permeabilized using 0.5% Triton-X100 for 15 minutes and blocked with 3% BSA for 1 hour. Coverslips were then probed for rickettsiae using rabbit anti-Rickettsia I1789 antibody diluted 1:1000, followed by the secondary antibody, Alexa 488 goat anti-rabbit (Invitrogen, A11008; 1:1000 dilution). For all immunofluorescence assays, samples with secondary antibody only served as a control for non-specific binding of the Alexa fluor antibodies.
For growth curves, cells were permeabilized using 0.5% Triton-X100 for 15 minutes and blocked with 3% BSA for 1 hour. Coverslips were then probed for rickettsiae using rabbit anti-Rickettsia I1789 antibody diluted 1:1000, followed by the secondary antibody, Alexa 488 goat anti-rabbit (Invitrogen, A11008; 1:1000 dilution). For all immunofluorescence assays, samples with secondary antibody only served as a control for non-specific binding of the Alexa fluor antibodies.
5
Immunohistochemistry on Paraffin Sections
6
Immunofluorescent Localization of aSMA in Murine Liver
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antigen retrieval of murine sections was achieved by microwaving in Tris–EDTA pH 9.0 for 15 min.
For immunofluorescent staining of aSMA, sections of murine liver were labelled with a monoclonal mouse antibody (Sigma A2547, clone 1A4, 1:1500 dilution, 1-h incubation at room temperature). Staining was visualized with donkey anti-mouse IgG (H and L) Alexa Fluor 555 conjugated secondary antibody (ThermoFisher Scientific), and sections mounted in VECTASHIELD HardSet Antifade Mounting Medium with DAPI (Vector Laboratories). Negative controls were performed using identical concentrations of species and isotype-matched non-immune immunoglobulin in place of primary antibody or omission of primary antibody.
10 × 20 objective fields centred on a central vein (in keeping with the pattern of damage of CCl4) were acquired using a Zeiss Axioplan II microscope and Photometrics CoolSNAP HQ2 camera, and separate TIFF images of each channel exported.
For immunofluorescent staining of aSMA, sections of murine liver were labelled with a monoclonal mouse antibody (Sigma A2547, clone 1A4, 1:1500 dilution, 1-h incubation at room temperature). Staining was visualized with donkey anti-mouse IgG (H and L) Alexa Fluor 555 conjugated secondary antibody (ThermoFisher Scientific), and sections mounted in VECTASHIELD HardSet Antifade Mounting Medium with DAPI (Vector Laboratories). Negative controls were performed using identical concentrations of species and isotype-matched non-immune immunoglobulin in place of primary antibody or omission of primary antibody.
10 × 20 objective fields centred on a central vein (in keeping with the pattern of damage of CCl4) were acquired using a Zeiss Axioplan II microscope and Photometrics CoolSNAP HQ2 camera, and separate TIFF images of each channel exported.
7
Immunofluorescence Imaging of Transfected HEK293 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
6 × 104 HEK293 cells per well were seeded into fibronectin-coated 8-chamber glass slides (ThermoFisher Scientific) and incubated for 24h. Cells were transfected with 4 ng indicated expression construct for 48h and fixed with 4% formaldehyde in PBS for 15 min. Fixed cells were washed three times with PBS (5 min each), blocked with 5% normal goat serum in PBS for 1h, and incubated with rabbit αFLAG antibody (Sigma-Aldrich, 10 μg/ml) in PBS/1% BSA for 1h. Cells were washed as before and incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (Jackson ImmunoResearch, 1:1000) in PBS/1% BSA for 1h. Cells were washed as before and coverslips mounted using VECTASHIELD HardSet Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA). Stained cells were imaged using a fluorescence microscope with Apotome (Zeiss, Oberkochen, Germany).
8
Immunoprecipitation and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies used in this study are reported in Supplementary Table S3 . Reagents and chemicals include: Ionomycin calcium salt (Sigma # I3909), Protein G-Agarose (Sigma #11243233001), Streptavidin Sepharose High Performance bead (Sigma #17-5113-01), Calmodulin Sepharose 4B (Sigma #17-0529-01), Dynabeads™ Protein G for Immunoprecipitation (Thermo Fisher Scientific #10004D), jetPRIME®, DNA and siRNA transfection reagent, Polyplus-transfection® reagent (VWR #89129-922), TRIzol™ Reagent (Thermo Fisher Scientific #15596018), Quantitect reverse transcriptase (Qiagen #205313), Xpert Protease inhibitor cocktail (GenDEPOT #P3100-100), Xpert Phosphatase inhibitor cocktail (GenDEPOT #P3200-020), Penicillin-Streptomycin (GenDEPOT #CA005-100), 10× PBS Buffer (GenDEPOT #P2100-100), DMEM, High Glucose with L-Glutamine (GenDEPOT #CM002-050), Opti-MEM™ Reduced Serum Medium (Thermo Fisher Scientific #31985070), FBS Opti-Gold, Us Origin (GenDEPOT #F0900-050), iTaq Universal SYBR® Green Supermix (Bio-Rad #1725124), Immun-Blot PVDF Membrane (Bio-Rad #1620177), TGX™ FastCast™ Acrylamide Kit (Bio-Rad #161-0173, #161-0175), Precision Plus Protein™ Dual Color Standards (Bio-Rad #1610394), Blotto, non-fat dry milk (Santa-Cruz # sc-2325), SuperSignal™ West Dura Extended Duration Substrate (Thermo Fisher Scientific #34076), VECTASHIELD HardSet Antifade Mounting Medium with DAPI (Vectorlabs #H-1500).
9
Perfusion and Sectioning of Murine Brains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
One week after the rabies injection, mice were deeply anesthetized and transcardial perfusion was performed with 4% paraformaldehyde (PFA). Brains were kept in 4% PFA overnight at 4°C and transferred to a 30% sucrose and 0.1% sodium azide in phosphate buffered saline (PBS) solution at 4°C. The bottom right side of the brain was cut approximately 1–2 mm deep to mark the injected hemisphere. Brains were then sectioned with a microtome with the thickness of 40 μm and kept in 0.1% sodium azide in PBS solution. Every fourth section was mounted such that the entire brain was screened at 120 μm intervals. Sections were then counterstained using either Neurotrace Blue 435/455 Blue Fluorescent Nissl Stain (Thermo Fisher Scientific) or Vectashield Hardset Antifade Mounting Medium with DAPI (Vector Laboratories).
10
Histological Examination of Kidney and Lung Tissues
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!