An improved procedure for whole-mount immunolocalization was adopted. In brief, 1 cm root pieces were mounted in 2% formaldehyde in microtubule-stabilizing buffer (MTSB) supplemented with 0.1% Triton X-100. Vacuum infiltration of explants, hydrophilization, cell wall digestion and membrane permeabilization were performed as described previously (Pasternak et al., 2015 (link)). After 3 h of blocking in BSA, samples were incubated with a GFP Tag antibody (1:200 dilution) coupled to Alexa Fluor 488 (Thermo Fisher Scientific) for 1 h at room temperature, followed by three washes and then mounted in Slow FadeTM Diamond anti-fade mountant for confocal microscopy.
Protocol detail
Whole-mount immunolocalization of GFP
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Alexa fluor 488
Antibody
Buffer
Cell wall
Confocal microscopy
Diamond
Digestion
Dilution
Formaldehyde
Membrane
Microtubule
Root
Triton x 100
Vacuum
Corresponding Organization : South China Botanical Garden
Other organizations : University of Chinese Academy of Sciences, Kazusa DNA Research Institute
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Variable analysis
independent variables
- Procedure for whole-mount immunolocalization
- Immunolocalization of GFP Tag
- Root pieces mounted in 2% formaldehyde in microtubule-stabilizing buffer (MTSB) supplemented with 0.1% Triton X-100
- Vacuum infiltration of explants, hydrophilization, cell wall digestion and membrane permeabilization
- Blocking in BSA for 3 h
- Incubation with GFP Tag antibody (1:200 dilution) coupled to Alexa Fluor 488 for 1 h at room temperature
- Three washes after antibody incubation
- Mounting in Slow Fade™ Diamond anti-fade mountant for confocal microscopy
- Positive control: Not specified
- Negative control: Not specified
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