Multitarget Immunohistochemistry for Confocal and Super-resolution Imaging of Plant Cell Wall Polysaccharides

Abstract

The plant cell wall (PCW) is a pecto-cellulosic extracellular matrix that envelopes the plant cell. By integrating extra-and intra-cellular cues, PCW mediates a plethora of essential physiological functions. Notably, it permits controlled and oriented tissue growth by tuning its local mechano-chemical properties. To refine our knowledge of these essential properties of PCW, we need an appropriate tool for the accurate observation of the native (in muro) structure of the cell wall components. The label-free techniques, such as AFM, EM, FTIR, and Raman microscopy, are used; however, they either do not have the chemical or spatial resolution. Immunolabeling with electron microscopy allows observation of the cell wall nanostructure, however, it is mostly limited to single and, less frequently, multiple labeling. Immunohistochemistry (IHC) is a versatile tool to analyze the distribution and localization of multiple biomolecules in the tissue. The subcellular resolution of chemical changes in the cell wall component can be observed with standard diffraction-limited optical microscopy. Furthermore, novel chemical imaging tools such as multicolor 3D dSTORM (Three-dimensional, direct Stochastic Optical Reconstruction Microscopy) nanoscopy makes it possible to resolve the native structure of the cell wall polymers with nanometer precision and in three dimensions.Here we present a protocol for preparing multi-target immunostaining of the PCW components taking as example Arabidopsis thaliana, Star fruit (Averrhoa carambola), and Maize thin tissue sections. This protocol is compatible with the standard confocal microscope, dSTORM nanoscope, and can also be implemented for other optical nanoscopy such as STED (Stimulated Emission Depletion Microscopy).The protocol can be adapted for any other subcellular compartments, plasma membrane, cytoplasmic, and intracellular organelles.

Multicolor 3D-dSTORM Reveals Native-State Ultrastructure of Polysaccharides' Network during Plant Cell Wall Assembly

“…Therefore, the buffer composition and optimal dye combination for multicolor 3D-dSTORM in biologically relevant applications are essential to maximizing the resolution ( Heilemann et al., 2008 ; Dempsey et al., 2011 ; Van De Linde et al., 2011 ; Lehmann et al., 2016 ; Nahidiazar et al., 2016 ). Previously we showed a two-color 3D-dSTORM on plant tissue sections using 2F4-GLOX (glucose oxidase-based, 2F4 is a T/Ca/S buffer, see Transparent Methods ) buffer ( Haas, et al., 2020a , 2020b ). Here we tested the OxEA buffer and defined the optimal conditions for three-color 3D-dSTORM observations.…”

“…Samples were rehydrated with successive bathing, 15 min each, 100% ethanol, 70% ethanol, 50% ethanol, 25% ethanol, 10% ethanol in 2F4 buffer, and finally 100% 2F4 buffer. A detailed protocol is available at Bio-Protocols (Haas, et al, 2020b). 2F4 (T/Ca/S) specific buffer: 20 mM Tris, 0.5 mM CaCl2, 150 mM NaCl at pH 8.…”

“…Immunolabelling All the immunolabelling steps were performed using a 2F4 buffer. For pectin extraction, before immunostaining, tissue sections were incubated with pectolyase (0.1% w/v, Sigma P3026) in the incubation buffer (0.2M Na2HPO4, 0.1M citric acid, pH4.8), as described in (Haas, et al, 2020b) and (Yang et al, 2016). Next, the free aldehyde groups were quenched using 50 mM NH4Cl (Sigma-Aldrich, catalog number: 254134) in 2F4 buffer for 15 min, and then washed 3 times with 2F4 buffer, each time 3-5 min.…”

“…Arabidopsis meristem was harvested from a plant grown on soil in the long-day condition in the growing chamber and harvested when the inflorescence was 1 cm long; flowers with visible sepals were removed, keeping all the closest flower buds as described in (Haas, et al, 2020b) and (Yang et al, 2016).…”

“…SMLM finally enables observing native-state polymeric and molecular ultrastructure with nanometer precision. Due to technical limitations (tissue-level imaging, strong autofluorescence), its application to plant science is sporadic ( Liesche et al., 2013 ; Eggert et al., 2014 ; Dong et al., 2015 ; Haas, et al., 2020b ; Haas, et al., 2020a ); however, its recent application is already changing the vision of the cell wall structure ( Haas, et al., 2020a ). Moreover, the biologically relevant three-color 3D nanoscopy implementation, even in simpler isolated cellular systems, is rare.…”