The amount of information from only one experiment is enormous. We can detect the presence of these cell wall polymers and know exactly how they are distributed in cells and tissues, and even determine their abundance. This method is designed in such a way that it can be applied to almost any kind of plant tissue from any species one may want to analyze.
Immunolocalization in plant tissues involves a lot of meticulous work, difficult to explain without actually seeing what happens. Before collecting the plant tissue sample, fill a glass vial with enough cold fixative solution to completely submerge all the samples. Then select the plant tissues to be analyzed and trim the samples to a size of no more than 16 square millimeters.
Immediately immerse the samples in the fixative solution. Glutaraldehyde and formaldehyde are both excellent fixatives, but both are hazardous and must be handled in the flow hood. Transfer the vial to a vacuum chamber and slowly apply vacuum.
When the pressure reaches negative 60 kilo pascals, the floating material in the vial will start to sink to the bottom. Maintain the pressure in the chamber at no more than negative 80 kilo pascals. The sinking process may take up to two hours.
After the samples have been in the vacuum chamber for two hours, slowly release the vacuum. Seal the glass vial and refrigerate it overnight at four degrees Celsius. After the overnight fixation, discard any samples that didn't sink to the bottom of the vial.
Wash the remaining samples with 25 millimolar PBS for 10 minutes and then wash them in 25 millimolar PIPES buffer for 20 minutes. It is extremely important to remove all floating samples because it's highly probable that the fixative has not penetrated the sample. Dehydrate the samples in an ethanol series, immersing the samples for 20 minutes at each ethanol concentration.
To perfuse the samples, add increasing concentrations of LR-white resin in ethanol, beginning with one part resin to three parts ethanol. At each concentration, incubate for 24 hours at four degrees Celsius. Replace the LR-white resin with fresh resin and incubate the samples for an additional 12 hours at four degrees Celsius.
Prepare the embedding gelatin capsules, selecting capsules that are slightly larger than the samples so that the samples can be completely enclosed in the resin. Label paper tags with pencil because ink will contaminate the resin. Apply one drop of fresh LR-white resin to the bottom of each capsule.
Place a sample in each capsule, then fill the capsules to maximum capacity with fresh resin. Put the cap on the capsule and press gently to seal it. To polymerize the resin, cure the capsules at 58 degrees Celsius until fully hardened around 24 to 48 hours.
After peeling the gelatin capsule from the LR-white resin, place the resin block under the stereo microscope. Using a sharp razor blade, trim the LR-white block at a 45 degree angle to the sample. Rotate the sample and make a second cut at a 90 degree angle to the first.
Continue cutting in the same pattern to form a pyramid with the apex of the pyramid directly above the sample's area of interest, then begin removing fine slices perpendicular to the major axis until the cutting surface reaches the sample. The target sample should ideally be enclosed in a trapezoid shape. Place the trimmed resin block in the ultra microtome.
Adjust the angle between the knife edge and the resin block. Cut sections from the block with a thickness between 200 and 700 nanometers. Use a wire loop to carefully lift some sampled sections from the microtome.
Place them in a drop of distilled deionized water on a glass slide. Evaporate the water by placing the slide on a hot plate at 50 degrees Celsius, then add a drop of stain to the sections. After 30 seconds, rinse off the stain and observe the slide under the microscope to verify the general state of the section and that the desired plant structure is visible.
Prepare a clean reaction slide by placing a drop of distilled deionized water in each well of the slide. Transfer one or two sample sections to each drop of water. Place the slide in a 10 centimeter square Petri dish, cover it and let it dry at 50 degrees Celsius.
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 distill deionized water for five 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 four 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 three to four 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. By pinpointing the localization of specific epitopes, the protocol enables characterization of the cell wall composition.
For example, 1, 5-arabinan is abundant in the cell wall of the developing Quercus suber anther. Scarcely esterified homogalacturonans are typically found at the root tip of Quercus suber embryo specifying mechanical properties of the organ. Xylogalacturonans are found in degenerating cells, such as the endosperm cell during the final stages of the Quercus suber acorn maturation.
AGP's epitopes recognized by JIM13 or JIM8 are found on structures related to reproduction, such as cell lines related to microgametogenesis in Arabidopsis thaliana and the stigmatic papillae and microbial of the basal angiosperm Trithuria submersa. Common mistakes in implementing this protocol are usually easy to detect and identify. When the washes are skipped or the reaction wells are allowed to dry, the secondary antibody will usually appear as a smear.
Aggregates of green fluorochrome will form if the unbound primary antibody is not properly washed away. Folding or detachment of the sections is usually related to poor adhesion due to the use of unclean slides or aggressive washing. Sample preparation is also critical.
The resin blocks should be free of cracks with a clearly visible pale yellow to light brown sample. Inefficiently embedded samples will show powdery white spots or areas. Keeping the sample size under eight millimeters is essential for penetration of the fixative solution.
Poorly fixed samples will appear dark brown or almost black. Excessive temperature can cause the resin to crack making sectioning of the sample impossible. The use of immunolocalization techniques open several research directions.
One can follow on with more specific techniques such as biochemical analysis of the cell wall or even proceed to a molecular approach. The results provided by this technique can help to understand the composition of the cell wall, an extremely complex structure very difficult to analyze by simple chemical analysis.
