Immunolocalization of Arabinogalactan Proteins and Pectins in Plant Tissue

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 the secondary antibody will be used to mark the location where the primary antibody connected to the sample after washing off the excess of primary ant....

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 t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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.)