Y4 was labelled with Alexa Fluor 488 dye (Life Technologies). COS-7 cells were transiently transfected with hKCNK9 or hKCNK3 and mcherry was co-transfected as a transfection control. After 36 h, cells were treated with 0.4 mg ml−1 Y4 either for 3 h at 4 °C or for 12 h at 30 °C. At the end of incubation, cells were washed with PBS, fixed with 4% paraformaldehyde, washed with PBS and mounted with ProLong Gold Antifade Mountant containing 4,6-diamidino-2-phenylindole (Life Technologies). Images were taken at × 63/1.4 with scan zoom 1.0. For co-localization analysis, COS-7 cells were co-transfected with red fluorescent protein-tagged EEA1 (Addgene). Images were taken at × 40/1.3 with scan zoom 1.0 and quantified using Imaris software (Bitplane). A minimum threshold of red and green channels was selected. The data represent images taken from 20 different areas (n=20).
Prolong gold antifade mountant containing 4 6 diamidino 2 phenylindole
Manufactured by Thermo Fisher Scientific
ProLong Gold Antifade Mountant is a ready-to-use, water-based mounting medium designed for fluorescence microscopy. It contains 4,6-diamidino-2-phenylindole (DAPI), a fluorescent dye that binds to DNA, allowing for the visualization of nuclei in fixed cells or tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab6672, by Abcam (2 mentions)
Alexa fluor 488 dye, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 conjugated donkey anti goat, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexafluor555 conjugated donkey anti goat antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
4 protocols using prolong gold antifade mountant containing 4 6 diamidino 2 phenylindole
1
Fluorescence Imaging of Ion Channel Localization
2
Fluorescent In Situ Hybridization of Helicobacter in Gastric Tissue
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Five micrometer human gastric tissue sections were deparaffinized and dehydrated in sequential washes of 50%, 80%, and 95% ethyl alcohol. After air drying, a hybridization cocktail containing 40% formamide, 0.1% SDS, 0.9 M NaCl, 20 mM Tris (pH 7.4), nuclease-free water, and 10 ng/μl fluorescently labeled probes (EUB338 probe: Cy3.5 5′-GCTGCCTCCCGTAGGAGT-3′ and Helicobacter-specific probe: Alexa488 5′-TCTCAGGCCGGATACCCGTCATAGCCT-3′; Eurofins) were added to the sections, and they were incubated in a humidified chamber at 37 °C overnight. The slides were washed in wash buffer containing 0.9 M NaCl and 25 mM Tris (pH 7.4) at 50 °C for 20 min. They were dipped in room temperature distilled water very briefly, dried at room temperature, and mounted with ProLong Gold Antifade Mountant containing 4′,6-diamidino-2-phenylindole (Life Technologies; P36935).
3
Immunofluorescent Staining of ACKR3 and IL6
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Cells were placed on glass slides, fixed in acetone at 25 °C for 5 min, and dried completely before staining. Cells were incubated overnight at 4 °C with either anti-ACKR3 (PA5-27077, Thermo Fisher Scientific) or anti-IL6 (ab6672; Abcam, Cambridge, UK) antibodies diluted 1:100 in phosphate-buffered saline (PBS). After washing 3 times with PBS, slides were incubated with Alexa Fluor 488-conjugated goat anti-rabbit or AlexaFluor555-conjugated donkey anti-goat (Thermo Fisher Scientific) antibodies, respectively, diluted 1:2000 in PBS, for 1 h at 25 °C. After incubation, nuclei were washed 3 times with PBS and counterstained using ProLong gold anti-fade mountant containing 4′,6-diamidino-2-phenylindole (Thermo Fisher Scientific).
4
Immunofluorescence Staining of Cathepsin F and IL-6
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Cells were placed on glass slides, fixed in acetone at room temperature (15–25 °C) for 5 min, and dried completely before staining. The cells were incubated overnight at 4 °C with an anti-cathepsin F antibody (PA5-87,925, Thermo Fisher Scientific) and anti-IL-6 antibody (ab6672, Abcam, Cambridge, UK) diluted 1:100 in phosphate-buffered saline (PBS). To confirm the specificity of the immunostaining, fetal bovine serum diluted to the same concentration was used instead of the primary antibody as a negative control. After washing three times with PBS, the slides were incubated with Alexa Fluor 488-conjugated goat anti-rabbit antibody and AlexaFluor555-conjugated donkey anti-goat antibody (Thermo Fisher Scientific) diluted 1:2000 in PBS for 1 h at room temperature. After incubation, nuclei were washed three times with PBS and counterstained for the visualization of nuclei using ProLong Gold Anti-fade Mountant containing 4′,6-diamidino-2-phenylindole (Thermo Fisher Scientific).
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