Gairdia parasites were iced for 30 min and pelleted at 700 x g for 7 min. The pellet was fixed in PME buffer (100 mM Piperazine-N,N’-bis (ethanesulfonic acid) (PIPES) pH 7.0, 5 mM EGTA, 10 mM MgSO4 supplemented with 1% paraformaldehyde (PFA) (Electron Microscopy Sciences, Hatfield, PA), 100 µM 3-maleimidobenzoic acid N-hydroxysuccinimide ester (Sigma-Aldrich), 100 µM ethylene glycol bis (succinimidyl succinate) (Pierce), and 0.025% Triton X-100 for 30 min at 37°C. Fixed cells were attached on polylysine coated coverslips. Cells were washed once in PME and permeabilized with 0.1% Triton X-100 in PME for 10 min. After two quick washes with PME, blocking was performed in PME supplemented with 1% bovine serum albumin, 0.1% NaN3, 100 mM lysine, 0.5% cold water fish skin gelatin (Sigma-Aldrich). Next, 1:200 diluted Alexa 647-conjugated anti-CWP1 antibody (Waterborne, New Orleans, LA) was incubated for 1 h. Cells were washed three times in PME plus 0.05% Triton X-100. Coverslips were mounted with ProLong Gold antifade plus 4’,6-diamidino-2-phenylinodole (DAPI; Molecular Probes). Images were acquired on a DeltaVision Elite microscope using a 60X, 1.4-numerical aperture objective with a PCO Edge sCMOS camera, and images were deconvolved using SoftWorx (API, Issaquah, WA).
Prolong gold antifade plus 4 6 diamidino 2 phenylinodole dapi
Manufactured by Thermo Fisher Scientific
ProLong Gold antifade plus 4',6-diamidino-2-phenylinodole (DAPI) is a laboratory reagent used to mount and preserve fluorescently labeled samples for microscopy. It contains an antifade agent to minimize photobleaching and DAPI, a fluorescent dye that binds to DNA, allowing visualization of cell nuclei.
Automatically generated - may contain errors
3 protocols using prolong gold antifade plus 4 6 diamidino 2 phenylinodole dapi
1
Immunofluorescence Staining of Giardia Parasites
2
Immunofluorescence Localization of CWP1 in Giardia
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Gairdia parasites were iced for 30 min and pelleted at 700 x g for 7 min. The pellet was fixed in PME buffer (100 mM Piperazine-N,N’-bis (ethanesulfonic acid) (PIPES) pH 7.0, 5 mM EGTA, 10 mM MgSO4 supplemented with 1% paraformaldehyde (PFA) (Electron Microscopy Sciences, Hatfield, PA), 100 μM 3-maleimidobenzoic acid N-hydroxysuccinimide ester (Sigma-Aldrich), 100 μM ethylene glycol bis (succinimidyl succinate) (Pierce), and 0.025% Triton X-100 for 30 min at 37°C. Fixed cells were attached on polylysine coated coverslips. Cells were washed once in PME and permeabilized with 0.1% Triton X-100 in PME for 10 min. After two quick washes with PME, blocking was performed in PME supplemented with 1% bovine serum albumin, 0.1% NaN3, 100 mM lysine, 0.5% cold water fish skin gelatin (Sigma-Aldrich). Next, 1:200 diluted Alexa 647-conjugated anti-CWP1 antibody (Waterborne, New Orleans, LA) was incubated for 1 h. Cells were washed three times in PME plus 0.05% Triton X-100. Coverslips were mounted with ProLong Gold antifade plus 4’,6-diamidino-2-phenylinodole (DAPI; Molecular Probes). Images were acquired on a DeltaVision Elite microscope using a 60X, 1.4-numerical aperture objective with a PCO Edge sCMOS camera, and images were deconvolved using SoftWorx (API, Issaquah, WA).
3
Microscopy of Giardia Parasites
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Gairdia parasites were iced for 30 min and pelleted at 700 x g for 7 min. The pellet was fixed in PME buffer (100 mM Piperazine-N,N'-bis (ethanesulfonic acid) (PIPES) pH 7.0, 5 mM EGTA, 10 mM MgSO4 supplemented with 1% paraformaldehyde (PFA) (Electron Microscopy Sciences, Hatfield, PA), 100 µM 3-maleimidobenzoic acid N-hydroxysuccinimide ester (Sigma-Aldrich), 100 µM ethylene glycol bis (succinimidyl succinate) (Pierce), and 0.025% Triton X-100 for 30 min at 37C. Fixed cells were attached on polylysine coated coverslips. Cells were washed once in PME and permeabilized with 0.1% Triton X-100 in PME for 10 min. After two quick washes with PME, blocking was performed in PME supplemented with 1% bovine serum albumin, 0.1% NaN3, 100 mM lysine, 0.5% cold water fish skin gelatin (Sigma-Aldrich). Next, 1:200 diluted Alexa 647-conjugated anti-CWP1 antibody (Waterborne, New Orleans, LA) was added to incubate for 1 h. Cells were washed three times in PME plus 0.05% Triton X-100. Coverslips were mounted with ProLong Gold antifade plus 4',6-diamidino-2-phenylinodole (DAPI; Molecular Probes). Images were acquired on a DeltaVision Elite microscope using a 60X, 1.4numerical aperture objective with a PCO Edge sCMOS camera, and images were deconvolved using SoftWorx (API, Issaquah, WA).
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