The following antibodies were used: rat anti-BrdU (1:2000; Accurate Chemical and Scientific, Westbury, NY, USA, cat. no. OBT0030G), mouse anti-NeuN (1:500; Millipore, cat. no. MAB377), mouse anti-β3 tubulin (1:5000; Promega, Madison, WI, USA) and mouse anti-GFAP (1:500; Sigma, cat. no. G3893). Sections were incubated overnight at 4°C with a cocktail of the primary antibodies. The rat anti-BrdU was detected with the donkey anti-rat IgG (H+L) labeled with Alexa Fluor® 488 (1:1000; Molecular Probes, Invitrogen, Paisley, UK, cat. no. A21208). The antibodies NeuN, β3-tubulin and GFAP were detected with the donkey anti-mouse IgG (H+L) labeled with Alexa Fluor® 594 (1:1000; Molecular Probes, cat. no. A21203). The sections labeled by double immunofluorescence (BrdU+NeuN, BrdU+β3-tubulin or BrdU+GFAP) were visualized with a confocal laser (spectral) scanning microscope (Leica TCS NT; Leica Microsystems) equipped with a 561 nm DPM laser (argon 30%) and a 40× objective (HCX PL APO CS 40.0x1.25 OIL UV). The emission filter settings were 500–550 nm for PMT2 (green) and 610–700 nm for PMT3 (red). Depending on the level of zoom used in each image, the XY voxel size was from 100 to 77 nm, approximately. Settings of light and brightness/contrast were adjusted by using the Leica LAS AF Lite imaging software.
Las af lite imaging software
Manufactured by Leica
LAS AF Lite is an imaging software developed by Leica. It provides basic functionalities for acquiring, processing, and analyzing microscope images. The software offers tools for image capture, annotation, and measurement, enabling users to perform essential tasks related to microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Hcx pl apo cs 40.0x1 25 oil uv, by Leica (2 mentions)
Tcs nt, by Leica (2 mentions)
Mouse anti gfap, by Merck Group (1 mentions)
Mouse anti β3 tubulin, by Promega (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Mouse anti neun, by Merck Group (1 mentions)
Af488 ctb, by Thermo Fisher Scientific (1 mentions)
Sp5 confocal fluorescence microscope, by Leica (1 mentions)
Nis elements imaging software 3, by Nikon (1 mentions)
3 protocols using las af lite imaging software
1
Double Immunofluorescence Labeling Protocol
2
Visualizing BCR Internalization and Lipid Rafts
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The surface BCRs were labeled and activated with AF546-F(ab′)2 goat anti–mouse IgM (5 µg/ml; Invitrogen) and lipid rafts by AF488-CTB (3 µg/ml; Invitrogen) at 4°C. After 5-min incubation with SLO (200 ng/ml) at 4°C, time-lapse images were acquired at 37°C using an SP5 confocal fluorescence microscope (63× oil objective 1.4 NA; Leica). Kymographs were constructed using Andor IQ (Andor Technology) to track the internalization of BCR and CTB over time. The colocalization rate (percentage of pixel overlap of two colors) of internalized BCRs with CTB at 10 min was determined using Leica LAS AF Lite imaging software. For each condition, more than eight cells were analyzed for each of three independent experiments.
3
Quantitative Analysis of Immunomarkers
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Positive (+) immunoreactive and immunofluorescent nuclei and cells that came into focus were manually counted. Representative counting frames from sections with immunoreactivity were evaluated using a standard optical microscope with the 40× objective (Nikon Instruments Europe B.V., Amstelveen, The Netherlands) coupled to the NIS-Elements Imaging Software 3.00 (Nikon). For immunofluorescent cell counting, the sections were visualized with a confocal laser (spectral) scanning microscope (Leica TCS NT; Leica Microsystems) equipped with a 561 nm DPM laser (argon 30%) and a 40× objective (HCX PL APO CS 40.0x1.25 OIL UV). The emission filter settings were 500-550 nm for PMT2 (green) and 610-700 nm for PMT3 (red). Depending on the level of zoom used in each image, the XY voxel size was from 100 to 77 nm, approximately. Settings of light and brightness/contrast were adjusted by using the Leica LAS AF Lite imaging software.
Phospho-H3+ and BrdU+ nucleus counts were performed in the subventricular zone (SVZ) of the lateral ventricles, while GFAP, Iba-1, iNOS, FosB, cleaved caspase-3 and PPARα+ cell counts were carried out in the whole striatum. Overall, quantification was expressed as the average number of cells per area (mm 2 ) for each experimental group.
Phospho-H3+ and BrdU+ nucleus counts were performed in the subventricular zone (SVZ) of the lateral ventricles, while GFAP, Iba-1, iNOS, FosB, cleaved caspase-3 and PPARα+ cell counts were carried out in the whole striatum. Overall, quantification was expressed as the average number of cells per area (mm 2 ) for each experimental group.
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