Abstract
This protocol was developed to study protein localisation within the vascular bundles of developing tomato fruit however, it can be applied to any resin embedded plant tissue. The vascular bundle is comprised of many different cells that all have unique properties. The mature sieve elements are enucleated and contain sieve plates that comprise of callose. This method has utilised these properties of the sieve element by combining immunohistochemistry for cell wall invertase with counterstaining of aniline blue for callose, DAPI for nucleus and cell structure is shown with the final staining of the cell wall using calcofluor white. It must be noted that when following this protocol, it is vital for the sections to be flat and fixed to the slide with gelatine so cover slip removal does not move the sample section. This protocol will be applicable to all plant tissues and provides additional evidence of the protein localisation within the cell by conducting a counterstaining procedure.
Keywords: Immunolocalisation, Microscopy, Staining, Phloem
Background
Immunolocalisation has long been a method used to study the localisation of proteins within tissue. This protocol focused on, not only localising the proteins of interest but also the molecular structures that were in surrounding tissue. It is believed that the mature sieve elements are enucleated and have abundant callose deposition within the sieve plate. Therefore, counterstaining procedures were applied in order to represent these biological phenomena. As the proteins of interest in this study were also thought to be localised within the apoplast further counterstaining was applied showing co-labelling of the protein and the cell wall.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Images sequences were imported into Adobe Bridge CS4 software before being loaded into Adobe Photoshop CS4 software and overlaid with opacity and intensity levels adjusted.
Notes
Gelatine coated slides need to be well coated to ensure stability of section on the microscope slide during removal of coverslip for optimal overlaying of images acquired.
Recipes
Acknowledgments
The condensed version of this method was originally published in Palmer et al., 2015. This work was collectively supported by the National Science Foundation of China (Grant number 30425043 to Y.L.R.) and Australia Research Council (ARC DP110104931 to Y.L.R. and DP120104148 to Y.L.R. and J.W.P.).
References
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