Resin-embedded Thin-section Immunohistochemistry Coupled with Triple Cellular Counterstaining

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.