For immunolocalisation of PMEs and HGs with different DMs, the roots were treated with Foc1 and Foc4 for 6, 12, 24 and 48 h and harvested at the same time points to minimise the effect of other factors. The protocol for fixation, embedding of samples and immunolabelling was carried out as described by Xu et al.56 . We labelled the sections with primary monoclonal antibodies PME, 2F4, LM19, LM20, JIM5 and JIM7. The anti-mouse lgG-FITC (F9006, Sigma) was used as secondary antibody for PME and 2F4 antibodies, and the anti-rat lgG-FITC (F6258, Sigma) was used for the other antibodies. Sections marked only with secondary antibodies were used as controls. At least three replicates were prepared for each antibody. Images were collected using a LSM 7 DUO laser scanning confocal microscope (ZEISS, Germany) and Olympus BH-2-FRCA microscope, and fluorescence signals were examined with LSM 7 DUO laser scanning confocal microscope (ZEISS, Germany) by the function “Histo” to determine an average fluorescence signal of a whole root section.
Lsm 7 duo laser scanning confocal microscope
Manufactured by Zeiss
Sourced in Germany
The LSM 7 DUO is a laser scanning confocal microscope manufactured by Zeiss. It is designed to capture high-resolution, three-dimensional images of samples using laser excitation and confocal optics.
Automatically generated - may contain errors
Lab products found in correlation
F9006, by Merck Group (1 mentions)
Bh 2 frca microscope, by Olympus (1 mentions)
Deadend fluorometric tunel kit, by Promega (1 mentions)
Click it edu imaging kit, by Thermo Fisher Scientific (1 mentions)
Hoechst dye, by Thermo Fisher Scientific (1 mentions)
Rabbit anti iba1, by Fujifilm (1 mentions)
Mouse anti iba1, by Merck Group (1 mentions)
4 protocols using lsm 7 duo laser scanning confocal microscope
1
Immunolocalization of Cell Wall Polymers
2
Anther Developmental Stages Analysis
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Anther developmental stages were confirmed by observing anther cross-sections with light microscopy. Preparation of anther sections and a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay used a Dead End Fluorometric TUNEL Kit (Promega, USA); these were performed as previously described (Li et al., 2006 (link); Luo et al., 2013 (link)). Fluorescein’s green fluorescence (TUNEL signal) and propidium iodide’s red fluorescence were imaged with 488 nm (excitation) and 520 nm (detection), and 488 nm (excitation) and 610 nm (detection), respectively, under a LSM 7 DUO laser scanning confocal microscope (Zeiss, Germany).
3
F-ara-EdU Labeling and Detection Protocol
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F-ara-EdU labeling and detection was performed as previously described with minor modifications57 (link). Seedlings grown in 1/2 MS were transferred to 1/2 MS media supplemented with 0.2 μM F-ara-EdU (Invitrogen Click-iT® EdU Imaging Kit). Whole seedlings or excised roots were fixed in 3.7% (v/v) formaldehyde solution for 1 day, treated with permeabilization buffer [0.5% (v/v) Triton-X in 1× PBS] for 20 min, and washed twice in wash buffer (3% BSA in 1× PBS). Then, the samples were incubated in click-iT reaction mixture (Invitrogen Click-iT® EdU Imaging Kit; 1× Click-iT® EdU reaction buffer, CuSO4, Alexa Fluor® azide, and 1× Click-iT® EdU buffer additive) for 30 min and washed twice in wash buffer, followed by a final wash in 1× PBS buffer. All procedures were performed at room temperature. The samples were mounted on glass slides using 30% glycerol and imaged immediately using a Zeiss LSM 7 DUO confocal laser scanning microscope (Carl Zeiss, Germany), with a 40× oil immersion objective. At least 15 seedlings from three independent experiments were analyzed.
4
Double-Labelling of Microglia Markers
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Double-labelling were performed for Iba-1 and cleaved caspase-8, Iba1 and CD16/32 and CD16/32 and cleaved caspase-8. Two different Iba1 antibodies were used: mouse anti-Iba-1 (Millipore) to co-localize with cleaved caspase-8 and rabbit anti-Iba-1 (Wako) to co-localize with CD16/32. Sections were blocked with PBS containing 1% appropriate serums (Vector Laboratories) for 1 hour. The slides were washed three times in PBS, then incubated overnight at 4°C with the two primary antibodies to be tested diluted in PBS containing 1% appropriate serums and 0.25% Triton X-100. Sections were incubated with the following secondary antibodies: a) mouse anti-Iba1: horse anti-mouse conjugated to Texas Red (1:200; Vector Laboratories), b) rabbit anti-Iba1 and rabbit anti-cleaved caspase-8: horse anti-rabbit conjugated to Fluorescein (1:200; Vector Laboratories), c) rat anti-CD16/32: chicken anti-rat conjugated to AlexaFluor 594 (1:200, Invitrogen). Incubation with secondary antibodies were for 2 hours at room temperature in the dark. Their addition was preceded by three 10-minute rinses in PBS. Nuclei were counterstained with Hoechst dye (1 μg/ml; Molecular Probes/Invitrogen). Fluorescence images were acquired using a Zeiss LSM 7 DUO confocal laser scanning microscope (Carl Zeiss Microscopy, Jena, Germany) and processed using the associated software package (ZEN 2010; Carl Zeiss Microscopy).
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