P. c. adami parasitemia was monitored by confocal microscopy to discriminate residual (strep +) from newly differentiated (strep-) infected and multiple infected RBCs and reticulocytes (CD71+). At peak infection, 2 μL of blood were fixed overnight with 1% paraformaldehyde, washed with PBS and stained with 1 μg/mL DAPI, anti-mouse CD71-FITC and APC-conjugated streptavidin for 30 min in the dark. RBCs were washed and pellet dissolved in 100 μL of PBS. Drops of RBCs suspension were settled on glass coverslips #1.5. RBCs were imaged by DIC and fluorescence microscopy (Nikon A1 confocal, plan Apochromat VC 60x, numerical aperture 1.4, λs oil immersion) and analyzed with NIS-Elements Viewer 4.20 imaging software (Nikon).
Plan apochromat vc 60x
Manufactured by Nikon
The Plan Apochromat VC 60x is a high-performance microscope objective lens produced by Nikon. It is designed to provide excellent optical performance with a numerical aperture of 1.40 and a working distance of 0.21 mm. The lens employs apochromatic correction and vibration compensation technology to deliver precise, high-contrast images.
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2 protocols using plan apochromat vc 60x
1
Confocal Microscopy of Malaria Parasites
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P. c. adami parasitemia was monitored by confocal microscopy to discriminate residual (strep +) from newly differentiated (strep-) infected and multiple infected RBCs and reticulocytes (CD71+). At peak infection, 2 μL of blood were fixed overnight with 1% paraformaldehyde, washed with PBS and stained with 1 μg/mL DAPI, anti-mouse CD71-FITC and APC-conjugated streptavidin for 30 min in the dark. RBCs were washed and pellet dissolved in 100 μL of PBS. Drops of RBCs suspension were settled on glass coverslips #1.5. RBCs were imaged by DIC and fluorescence microscopy (Nikon A1 confocal, plan Apochromat VC 60x, numerical aperture 1.4, λs oil immersion) and analyzed with NIS-Elements Viewer 4.20 imaging software (Nikon).
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P. c. adami parasitemia was monitored by confocal microscopy to discriminate residual (strep +) from newly differentiated (strep-) infected and multiple infected RBCs and reticulocytes (CD71+). At peak infection, 2 μL of blood were fixed overnight with 1% paraformaldehyde, washed with PBS and stained with 1 μg/mL DAPI, anti-mouse CD71-FITC and APC-conjugated streptavidin for 30 min in the dark. RBCs were washed and pellet dissolved in 100 μL of PBS. Drops of RBCs suspension were settled on glass coverslips #1.5. RBCs were imaged by DIC and fluorescence microscopy (Nikon A1 confocal, plan Apochromat VC 60x, numerical aperture 1.4, λs oil immersion) and analyzed with NIS-Elements Viewer 4.20 imaging software (Nikon).
2
Immunofluorescence Analysis of Mouse Brain
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Under terminal (ketamine-xylazine) anaesthesia mice were transcardially perfused with 4% paraformaldehyde (PFA) and brains were dissected. Brain samples were post-fixed, cryoprotected for 24h and 25 µm thick coronal sections were cut, using a sledge microtome (Leica, Germany).
Immunostaining was performed on free-floating brain sections, blocked with 5% normal donkey serum (Jackson ImmunoResearch Europe Ltd, Ely, Cambridgeshire, UK). The following primary antibodies were used: rabbit anti-P2Y12R (1:500, #55043AS AnaSpec), chicken anti-GFP-tag (1:500, #A10262 Invitrogen), rat anti-CD206 (1:200, #MCA2235, AbD Serotec) and biotinylated tomato lectin (1:500, #016-470-084, Jackson ImmunoResearch), streptavidin A594 (1:500, #S11227, Invitrogen).
Incubation was followed by washing in TBS and PB, then sections were mounted on glass slides with Aqua-Poly/Mount (Polysciences). Immunofluorescence was analyzed using a Nikon Eclipse Ti-E inverted microscope (Nikon Instruments Europe B.V., Amsterdam, The Netherlands), with a CFI Plan Apochromat VC 60X oil immersion objective (NA 1.4) or a Plan Apochromat VC 20X objective (NA 0.75) and an A1R laser confocal system. The following lasers were used: 405, 488, 561 and 647 nm (CVI Melles Griot). Scanning was done in line serial mode. Image stacks were obtained with NIS-Elements AR 5.00.00 software.
Immunostaining was performed on free-floating brain sections, blocked with 5% normal donkey serum (Jackson ImmunoResearch Europe Ltd, Ely, Cambridgeshire, UK). The following primary antibodies were used: rabbit anti-P2Y12R (1:500, #55043AS AnaSpec), chicken anti-GFP-tag (1:500, #A10262 Invitrogen), rat anti-CD206 (1:200, #MCA2235, AbD Serotec) and biotinylated tomato lectin (1:500, #016-470-084, Jackson ImmunoResearch), streptavidin A594 (1:500, #S11227, Invitrogen).
Incubation was followed by washing in TBS and PB, then sections were mounted on glass slides with Aqua-Poly/Mount (Polysciences). Immunofluorescence was analyzed using a Nikon Eclipse Ti-E inverted microscope (Nikon Instruments Europe B.V., Amsterdam, The Netherlands), with a CFI Plan Apochromat VC 60X oil immersion objective (NA 1.4) or a Plan Apochromat VC 20X objective (NA 0.75) and an A1R laser confocal system. The following lasers were used: 405, 488, 561 and 647 nm (CVI Melles Griot). Scanning was done in line serial mode. Image stacks were obtained with NIS-Elements AR 5.00.00 software.
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