To detect stage-specific TRAP protein expression, smears of each stage were made on positively charged slides and stored at −80 °C as previously described [29 (link)]. Polyclonal antibodies were produced by immunizing rabbits with TRAP peptides (Table 2 ) predicted as surface-exposed moieties (GenScript, Piscataway, NJ, USA). Slides were stained for IFA as previously described [16 (link)]. A secondary conjugate of goat-anti-rabbit IgG Alexa Fluor 555 (Thermo Fisher Scientific) was used to detect specific antibody reactivity. ProLong Gold antifade reagent with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Thermo Fisher Scientific) was used to stain nuclei. A Leica microscope (Buffalo Grove, IL, USA) was used to examine antibody reactivity. Since HAP2 is expressed by B. bovis sexual stages but not blood stages, we used rabbit anti-HAP2 antibody as controls [14 (link)] to distinguish B. bovis sexual stages from blood stages.
Prolong gold antifade reagent with 4 6 diamidino 2 phenylindole dihydrochloride dapi
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
ProLong Gold antifade reagent with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) is a mounting medium used to preserve and protect fluorescent signals in microscopy samples. It contains an antifade agent to reduce photobleaching and DAPI, a fluorescent dye that binds to DNA, allowing for visualization of cell nuclei.
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4 protocols using prolong gold antifade reagent with 4 6 diamidino 2 phenylindole dihydrochloride dapi
1
Immunolocalization of TRAP Protein in Babesia
2
Immunofluorescent Staining Protocol for NF-H
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Sections for immunofluorescent staining were bleached in Autofluorescence Eliminator Reagent (Millipore) according to the manufacturer's guidelines. Sections were then pre-incubated with 5% fetal calf serum diluted in phosphate-buffered saline (PBS) containing 0.25% Triton (PBS-T). Sections were incubated overnight with anti-NF-H antibody (clone TA51, 1:50, Millipore). On the following day, sections were rinsed in PBS-T for 10 min and incubated with Alexa-488-conjugated donkey anti-rat (1:500, Invitrogen) for 2 h at room temperature. Finally, sections were rinsed 3 × in PBS, 1 × in TBS, before being mounted in ProLong Gold Antifade Reagent with 4′,6-diamidino-2′-phenylindole dihydrochloride (DAPI) (Thermo Fisher Scientific).
3
Lumogallion Staining for Aluminum Detection in Cells
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Rehydrated agar-cell sections were stained by fully immersing slides in 250.0 mL 100 μM lumogallion (TCI Europe N.V. Belgium) buffered in 50 mM PIPES, pH 7.4. For autofluorescence analyses of THP-1 cells, sections were placed into the same PIPES-buffer in the absence of added lumogallion. All sections were covered and incubated at ambient temperature in the dark for 24 h. lumogallion stained cell sections were subsequently rinsed by agitation in 50 mM PIPES, pH 7.4 for 2 min, prior to rinsing for 30 s in ultrapure water to remove excess buffer. Sections for autofluorescence were rinsed in ultrapure water only. The latter was performed to prevent precipitation of PIPES upon the drying of cell sections. Sections were finally air dried and mounted using ProLong® Gold Antifade Reagent with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Life Technologies, UK) prior to storing horizontally for 24 h at 4 °C.
4
Fluorescent Staining of Cells and Tissues
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Lumogallion [4-chloro-3-(2,4-dihydroxyphenylazo)-2-hydroxybenzene-1-sulphonic acid] (Tokyo Chemical Industry, Oxford, UK) was prepared at 1 mM via dilution into 0.22 μm filtered 50 mM PIPES buffer, pH 7.4. Deparaffinised and rehydrated THP-1 cell or brain serial sections were stained away from light for 45 min at ambient temperature with either lumogallion or 50 mM PIPES buffer only, of which the latter acted as a control for autofluorescence. Following staining, sections were subsequently washed six times with 200 μL aliquots of 50 mM PIPES buffer, prior to a 30 s rinse in ultrapure water. Mounting of stained sections was performed using glass coverslips using the aqueous mounting medium Fluoromount™ for all brain tissue sections and the aqueous mounting medium, ProLong® Gold Antifade Reagent with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Life Technologies, Fisher Scientific, Loughborough, UK) for THP-1 cell sections. The latter was used to highlight THP-1 cell nuclei. All sections were incubated overnight at 4 °C away from light allowing for the respective polymers to harden, prior to analysis via fluorescence microscopy.
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