Rainbow trout satellite cells were passaged into 12-well plates (at a concentration of 2.5 × 105 cells per well) 24 h prior to infection by SDV. At 3 days post-infection, cells were fixed with a mixture of ethanol and acetone [1:1 (v/v)] at −20 °C for 15 min. Antigen detection was performed by incubation with anti-E2 mAb and anti-desmin pAb (Dako) diluted 1:10 000 and 1:200, respectively, in PBS1x/0.05% Tween 20 for 45 min at room temperature. Cells were then washed three times, incubated with Alexa Fluor 488- and 594-conjugated anti-mouse or anti-rabbit immunoglobulins (Invitrogen), respectively, for 45 min at room temperature, washed again and mounted in Dapi Fluoromount G medium (Southern Biotech). Finally, cell monolayers were visualized directly with a UV-light microscope (Carl Zeiss).
Dapi fluoromount g medium
Manufactured by Southern Biotech
DAPI Fluoromount-G medium is a mounting medium designed for the preservation and visualization of fluorescent-labeled samples. It contains 4',6-diamidino-2-phenylindole (DAPI), a fluorescent dye that binds to the DNA in cell nuclei, allowing for the staining and observation of cellular structures.
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Lab products found in correlation
Vybrant dii cell labeling solution, by Thermo Fisher Scientific (2 mentions)
Uv light microscope, by Zeiss (1 mentions)
Fluorescently labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
Polylysine coated slides, by Thermo Fisher Scientific (1 mentions)
Roti block, by Carl Roth (1 mentions)
Whatman, by Cytiva (1 mentions)
Protran, by Cytiva (1 mentions)
Vybrant dii cell labelling solution, by Thermo Fisher Scientific (1 mentions)
C2 confocal microscope, by Nikon (1 mentions)
Anti γ tubulin antibody, by Merck Group (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
9 protocols using dapi fluoromount g medium
1
Immunofluorescence Detection of Viral Antigens
2
Apoptosis Detection in Cardiac Tissue
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TUNEL staining was performed following the manufacturer’s protocol of In Situ Cell Death Detection Kit (TMR red) (Roche, 12156792910). Briefly, after immunofluorescent staining with anti-cTnT antibody (1:200, Thermo Fisher Scientific, MS-295-P), heart tissue sections or cell samples were permeabilized with permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min on ice and washed with PBS twice. Samples were then stained with TUNEL reaction mixture for 1 h at 37 °C. After washing with PBS three times for 5 min each, the samples were mounted with DAPI Fluoromount-G medium (SouthernBiotech, 0100-20) for imaging.
3
Histological reconstruction of electrode implants
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Electrodes were labeled with a drop of Vybrant DiI cell-labeling solution (Thermo Fisher Scientific), applied with a needle tip before implantation, allowing postmortem reconstruction of the implant location. In order to do so, rats were perfused transcardially with saline and 10% formalin, their brains extracted and placed in 30% sucrose for 3–5 d. Brains were then frozen; coronal sections were cut around the implant location using a sliding microtome, mounted on glass slides in DAPI Fluoromount-G medium (Southern Biotech) and a coverslip was applied. Epifluorescence microscopy was used to visualize DiI and DAPI. Figure 1 shows histologic reconstruction of electrode placement in pPC for two representative animals.
4
Immunostaining of Early Embryos
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Immunostaining of early embryos was done essentially according to the ‘freeze-crack’ protocol. Gravid worms were dissected onto poly-lysine-coated slides (Thermo Fisher Scientific) and frozen in liquid nitrogen, followed by incubation in methanol at −20°C for 20 min and in acetone at −20°C for 20 min. After rehydration in PBS and blocking in 5% BSA, embryos were incubated with primary antibody overnight at 4°C. Incubation with the fluorescently labeled secondary antibodies (Life Technologies) was done at room temperature for 1 hr. Embryos were then mounted in DAPI Fluoromount G medium (SouthernBiotech). For quantification of RAD-51 positive embryos, all embryos on a slide where counted, irrespective of developmental stage, by focusing through embryos to categorize into RAD-51 foci positive or negative. For western blotting, purified proteins and worm lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes (Whatman, Protran). Membranes were blocked in 3% milk solution and incubated with the primary antibodies overnight at 4°C in RotiBlock (Carl Roth). Incubation with fluorescently labeled secondary antibodies was done at room temperature, before detection of signals using the Li-Cor Odyssey scanner.
5
Histological Reconstruction of Electrode Placement
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Electrodes were labelled with a drop of Vybrant® DiI cell‐labelling solution (www.thermofisher.com ), applied with a needle tip before implantation, allowing post mortem reconstruction of the implant location. Accordingly, rats were perfused trans‐cardially with saline and 10% formalin, their brains extracted and placed in 30% sucrose for 3–5 days. Brains were then frozen; coronal sections were cut around the implant location using a sliding microtome, mounted on glass slides in DAPI Fluoromount‐G medium (www.southernbiotech.com ) and a cover slip was applied. Epifluorescence microscopy was used to visualize 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (DiI) and 4′,6‐diamidino‐2‐phenylindole (DAPI). Figure 1 shows histological reconstruction of electrode placement in pPC for two representative animals.
6
Immunofluorescence Assay of Bdnf Neurons
7
Immunostaining of Embryonic γ-Tubulin
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Embryos were fixed with 4% PFA overnight at 4°C and dehydrated in absolute methanol for at least 20 min at −20°C. After washing thrice with 1 × PBST, the embryos were then permeabilized in −20°C acetone for 8 min. After washing, the embryos were blocked with blocking buffer (2% lamb serum, 0.1% dimethyl sulfoxide, 0.1% bovine serum albumin, and 0.2% Triton-X100 in PBS) for 1 h at room temperature. The embryos were treated with anti-γ-tubulin antibody (Sigma) at a dilution of 1:1000 in blocking buffer overnight at 4°C. After washing with PBST for 30 min three to five times at room temperature, the embryos were incubated in fluorescein-conjugated secondary goat antirabbit antibody (Alexa-fluor 555, 1:500, Life Technology, United States) at 1:1000 dilution for 2 h at room temperature. The embryos were then washed eight times in PBST for 5 min each. The whole embryos were mounted in a confocal dish with DAPI Fluoromount-G medium (Southern Biotech) and incubated for 5 min prior to imaging.
8
EBV Glycoprotein Expression Visualization
9
Electrode Implantation and Histological Reconstruction
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