Cells were seeded in poly-d -lysine-coated chamber slides at a density of 2 × 103 cells/well and grown until they reached 70 to 80% confluence for transfection. Twenty-four hours after transfection, cells were fixed for 10 min in 4% formaldehyde and then permeabilized in 0.5% Triton X-100 in PBS at room temperature. Slides were blocked in 10% goat serum (ThermoFisher Scientific) at 37°C for 1 h, followed by incubation with anti-FLAG, anti-TDP43, or anti-ubiquitin P4D1 antibody, as indicated in the figures, at a 1:1,000 dilution in 1× PBS containing 5% goat serum and 0.1% Tween 20 for 2 h at 37°C. Slides were washed three times with 1× PBS containing 0.1% Tween 20 at room temperature and then incubated with secondary antibodies (goat anti-mouse antibody–Alexa Fluor 488 and goat anti-rabbit antibody–Alexa Fluor 546) at a 1:500 dilution for 1 h. Finally, the slides were washed three times with PBS containing 0.1% Tween 20 and once with PBS. The slides were then mounted with 4′,6-diamidino-2-phenylindole (DAPI)-containing Vectashield H-1200 mounting medium (Vector Laboratories). Aggregates were quantified by using a Nikon A1T-A1 confocal system with a Nikon Eclipse Ti inverted microscope and Nikon Instrument Software (NIS) elements AR-3.2 imaging software (Nikon Instruments, Melville, NY).
Vectashield mounting medium h 1200
Manufactured by Vector Laboratories
Sourced in United States
Vectashield mounting medium (H-1200) is a glycerol-based aqueous mounting medium designed for use in fluorescence microscopy. It is formulated to reduce photobleaching of fluorescent dyes and proteins, allowing for improved fluorescence preservation and prolonged imaging.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 488 dextran conjugate, by Thermo Fisher Scientific (2 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Eclipse ti inverted microscope, by Nikon (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Anti p53 antibody, by Cell Signaling Technology (1 mentions)
Ti e microscope, by Nikon (1 mentions)
Iba 1, by Proteintech (1 mentions)
Cle caspase 3, by Cell Signaling Technology (1 mentions)
Parp1, by Proteintech (1 mentions)
Anti neun, by Merck Group (1 mentions)
Caspase 1, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 594 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Actingreen 488 readyprobes reagent, by Thermo Fisher Scientific (1 mentions)
N cadherin, by Thermo Fisher Scientific (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Confocal software v 2, by Leica (1 mentions)
Tcs sp2 a0bs confocal system, by Leica (1 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Tcs sp8, by Leica (1 mentions)
12 protocols using vectashield mounting medium h 1200
1
Immunofluorescence Imaging of Protein Aggregates
2
Quantitative Telomere Length Measurement
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Telomere length was measured by qFISH using a Cy3-labeled LL(CCCTAA)3 PNA telomeric probe (Eurogentec, Liège, Belgium). Following hybridization, slides were washed three times with PBS-0.1% Tween for 10 min at 60°C and dehydrated through an ethanol series (70, 90 and 100%, 5 min each). Slides were then counterstained and mounted in Vectashield H-1200 mounting medium (Vector Laboratories, Burlingame, CA). Digital images were acquired with a Leica DM2500/TCS SPE confocal microscope and camera set-up. Telomere signals were captured with the same exposure time in all samples, from at least 20–30 nuclei per group, and were quantified using the TFL-Telo software (version 2) (Vancouver, Canada).
3
Immunofluorescence Staining of Fibronectin and TKT
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Immunofluorescent staining was performed similarly as previously described 16 (link). Briefly, cells were grown on coverslips coated with 1% poly-L-lysine, fixed in 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100 in PBS. After washing, cells were blocked with FBS and incubated with anti-fibronectin or anti-TKT primary antibodies overnight. After washing, cells were incubated with FITC- or PE-conjugated secondary antibodies, and mounted with Vectashield H-1200 Mounting Medium (Vector Laboratories, Burlingame, CA). The immunofluorescence was visualized under a Zeiss fluorescence microscope (Carl Zeiss, Thornword, NY).
4
Immunofluorescence Assay of p53 Expression
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Cells were spread onto coverslips and treated with or without DFO for different times. After washing twice in PBS, the cells were fixed with 3% paraformaldehyde–2% sucrose for 10 min at room temperature and permeabilized with 1% NP40 in PBS for 5 min at room temperature. After blocking in 5% BSA, the coverslips were incubated with an anti-p53 antibody (1:500, 9282, Cell Signaling Technology, MA, USA) overnight at 4 °C in a humid chamber and then with Alexa Fluor 568-conjugated anti-rabbit IgG secondary antibody (Life technologies corporation, Frederick, MD, USA) for 1 h at room temperature in the dark. Slides were mounted in VECTASHIELD mounting medium (H-1200, Vector Labs, Newark, CA, USA). Images were captured on a Nikon Ti-E microscope using equal exposure times for all images.
5
Multimodal Immunofluorescence Staining Protocol
6
Histological Analysis of Mouse Testes
7
Immunofluorescent Staining of Endothelial Tight Junctions
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Endothelial cells from the in vitro BBB model were fixed with 3% paraformaldehyde in PBS for 10 min at room temperature and treated with PBS containing 0.1% Triton X-100 for 10 min after being washed with PBS containing Mg2+ and Ca2+ to permeabilize the cells. After fixation, the cells were incubated with 3% BSA in PBS for 1 h to block the nonspecific binding of antibodies. Subsequently, the endothelial cells were incubated with rabbit polyclonal antibodies against Claudin-5 (Z43.JK, Invitrogen, CA, USA), Occludin (ZMD.467, Invitrogen), ZO-1 (ZMD.437, Invitrogen) and N-cadherin (3B9, Invitrogen) at 37 °C for 1 h. After washing with PBS containing Mg2+ and Ca2+, they were incubated with Alexa Fluor 594-conjugated anti-rabbit IgG (Invitrogen) for 1 h at 37 °C. Actin was stained with ActinGreen 488 ReadyProbes Reagent (R37110, Molecular Probes). The stained cells were then washed in PBS without Mg2+ and Ca2+ and were mounted in VECTASHIELD Mounting Medium (H-1200, Vector Laboratories, CA, USA) for observation under a confocal microscope (FluoView FV1000, Olympus, Tokyo, Japan).
8
Immunofluorescence Staining of P. aeruginosa
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The cells were fixed with 4% paraformaldehyde for 15 min at room temperature. The fixed cells were washed with PBS three times and then permeabilized with 0.3% Triton X-100 for 20 min. Furthermore, the cells were blocked in 5% ordinary goat serum for 1–2 h at room temperature. Rabbit antikeratin 14 and PAS antibodies (Thermo, Rockford, USA), and rabbit anti-P. aeruginosa (home-made antibody; 1:200) were added to the cells and incubated overnight at 4°C. After removing the primary antibody, the cells were washed three times using PBS. Alexa Fluor 594-labeled antibody (green) and Alexa Fluor 594-labeled antibody (red; Jackson Immuno Research Laboratories, West Grove, PA, USA; 1:500) were added to the cells as fluorescent secondary antibodies to detect primary antibodies. The fluorescent secondary antibodies were removed and washed three times with PBS, and the membrane was fixed on a glass slide by Vectashield Mounting Medium (H-1200, Vector Laboratories, Burlingame, CA) with 4’ 6-diamidino-2-phenylindole (DAPI). Colocalization images were acquired by the Leica TCS SP2 A0BS confocal system and analyzed by the Leica Confocal Software v. 2.6.1 (Leica, Germany).
9
Retrograde Labeling of Motor Neurons
10
Retrograde Labeling of Motor Neurons
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Motor neurons were back filled with Alexa 488-dextran conjugate (Invitrogen). The fluorescent dextran was mixed with a small volume of distilled water and the viscous paste was loaded into the tip of a thin insect pin. Larvae grown at different temperatures were anesthetized with 0.1% MS222 and stabbed with the loaded needle through the skin and into the right ventral musculature. Larvae were kept in anesthetic for 15 min followed by washes with 10% MMR where they were allowed to recover for 2 h. Then larvae were anesthetized again and fixed with 2% glutaraldehyde in phosphate buffer for 30 min at room temperature. Fixed samples were dissected to expose the spinal cord by removing the skin and muscle on the right side. Exposed spinal cords were mounted in Vectashield mounting medium H1200 (Vector Labs) and confocally imaged with a 20X objective with a Nikon C2 microscope. Labeled cells were counted from at least 10 larvae per treatment.
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