Immunolocalization of Arabinogalactan Proteins and Pectins in Plant Tissue
Published: April 30, 2024
Abstract
Source: Costa, M., et al. Fluorescent Immunolocalization of Arabinogalactan Proteins and Pectins in the Cell Wall of Plant Tissues. J. Vis. Exp. (2021).
This video demonstrates an immunostaining technique to localize arabinogalactan proteins (AGPs) and pectins in plant tissues. Fixed and resin-embedded plant tissue sections are stained with AGP- and pectin-specific primary and fluorescent secondary antibodies and a cellulose-binding fluorescent dye to reveal their tissue-specific colocalization in the cell wall.
Protocol
1. Immunolocalization NOTE: The fluorescent immunolocalization procedure relies on sequential use of two antibodies. The primary antibody is raised against a specific target antigen. The secondary antibody is raised specifically against the primary antibody and for fluorescent techniques is conjugated to a fluorophore (FITC [fluorescein isothiocyanate] in this specific protocol). The primary antibody will be used to detect the target antigen in the sample, and t…
Representative Results
Figure 1. (A) Schematic representation of the block trimming sequence for preparing the sample (Yellow block) for sectioning with the ultramicrotome. Firstly, the gelatin capsule is removed (1). Then they are first trimmed at a 40° to 45° angle to the sides of the capsule tangentially to the sample (2), a second cut is made at 90° of the first (3) followed by a third (4) and fourth…
Divulgazioni
The authors have nothing to disclose.
Materials
| 8 wells Glass reaction slides | Marinfeld | MARI1216750 | other brands may be used |
| Anti-Rat IgG (wole molecule)-FITC antibody produce in GOAT | Sigma-Aldrich | F6258 | |
| cover slips, 24 mm x 50 mm | Marinfeld | MARI0100222 | The cover slip should cover all the wells. Other brands may be used |
| ddH2O | na | na | |
| Ethanol absolute | na | na | |
| Fluorescent brightner 28 | Sigma-Aldrich | F-6259 | |
| Non fat dry milk | Nestlé | na | any non fat dry milk is adequate |
| Oven | na | na | generic laboratory oven |
| Rat generated Monoclonal Anti-Body | Plant probes | na | Several antibodies that recognize cell wall components are available at both the Complex Carbohydrate research center (CCRC, Georgia University USA) and Plant Probes (Paul Knox Cell Wall Lab, at Leeds University UK). A short list of some commonly used MABS and where they can be purchased is presented in Supplemental Table 1 |
| upright epifluorescence microscope with UV and FITC fluorescence filters | Leica Mycrosistems | DMLb | |
| vaccum chamber | na | na | |
| vaccum pump | na | na | |
| Vectashield | vecta Labs | T-1000 | Other anti-fade may be used. Please do check for compatibility with FITC and the Fluorescente brightner 28. (Note: for a non-commercial alternative, (see Jonhson et al 198218) An antifade medium can be made by mixing 25 mg/mL of 1,4-Diazobicyclo-(2,2,2)octane (DABCO) in 9:1 (v/v) glycerol to 1xPBS. Adjust pH to 8.6 with diluted HCl.) |
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Citazione di questo articolo
Immunolocalization of Arabinogalactan Proteins and Pectins in Plant Tissue. J. Vis. Exp. (Pending Publication), e22117, doi: (2024).