Immunolocalization of Arabinogalactan Proteins and Pectins in Plant Tissue

To prepare an incubation chamber for the reaction slides, place some dampened paper towels at the bottom of a pipette tips box and wrap the box with aluminum foil. Transfer the slides from the box to the incubation chamber. Pipette 50 microliters of blocking solution into each well. After incubating the slide for 10 minutes, remove the blocking solution. Then, wash all the wells twice with PBS for 10 minutes each time. After preparing the primary antibody solutions as described in the manuscript, perform a final wash of the wells with distilled, deionized water for 5 minutes.

Pipette the primary antibody solution into the reaction wells. Then, pipette the blocking solution into the control wells. Close the incubation chamber. Let it stand for two hours at room temperature, and then refrigerate it overnight at 4 degrees Celsius. Prepare a 1% solution of the secondary antibody in blocking solution. Approximately 40 microliters per well will be needed.

Cover the solution with aluminum foil. Wash the wells of the reaction slide twice with PBS, and once with distilled, deionized water for 10 minutes each time. Ensure that no blocking solution or deposits remain in the wells. Pipette the secondary antibody solution into all the wells. Incubate the slides in the dark at room temperature for 3 to 4 hours.

Again, wash the wells of the reaction slide twice with PBS, and once with distilled, deionized water. Then add a drop of calcofluor-white to each well. Without washing, add a drop of mounting medium to each well, and cover each well with a coverslip. Observe the sample sections in the reaction slide using a fluorescence microscope.

For each observation, use a UV filter to detect cell walls stained by the calcofluor and a FITC filter to detect immunolocalization. For a better visualization of the results, use ImageJ or a similar image analysis program to merge each UV filter image with the corresponding FITC filter image.