title: Protocol: an improved and universal procedure for whole-mount immunolocalization in plants.
abstract: Rapid advances in microscopy have boosted research on cell biology. However sample preparation enabling excellent reproducible tissue preservation and cell labeling for in depth microscopic analysis of inner cell layers, tissues and organs still represents a major challenge for immunolocalization studies. Here we describe a protocol for whole-mount immunolocalization of proteins which is applicable to a wide range of plant species. The protocol is improved and robust for optimal sample fixation, tissue clearing and multi-protein staining procedures and can be used in combination with simultaneous detection of specific sequences of nucleic acids. In addition, cell wall and nucleus labelling can be implemented in the protocol, thereby allowing a detailed analysis of morphology and gene expression patterns with single-cell resolution. Besides enabling accurate, high resolution and reproducible protein detection in expression and localization studies, the procedure takes a single working day to complete without the need for robotic equipment.
authors.affiliation: Faculty of Biology, Institute of Biology II/Molecular Plant Physiology, University of Freiburg, Freiburg, Germany.
authors.forename: Taras
authors.initials: T
authors.lastname: Pasternak
authors.affiliation: Faculty of Biology, Institute of Biology II/Molecular Plant Physiology, University of Freiburg, Freiburg, Germany.
authors.forename: Olaf
authors.initials: O
authors.lastname: Tietz
authors.affiliation: Faculty of Biology, Institute of Biology II/Molecular Plant Physiology, University of Freiburg, Freiburg, Germany.
authors.forename: Katja
authors.initials: K
authors.lastname: Rapp
authors.affiliation: Department of Agronomy, Food, Natural Resources, Animals and Environment, DAFNAE, University of Padova, Agripolis, Viale dell'Università, 35020 Legnaro, Padova Italy.
authors.forename: Maura
authors.initials: M
authors.lastname: Begheldo
authors.affiliation: BIOSS Centre for Biological Signaling Studies, University of Freiburg, Freiburg, Germany ; Center for Biological Systems Analysis, University of Freiburg, Freiburg, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Nitschke
authors.affiliation: Department of Agronomy, Food, Natural Resources, Animals and Environment, DAFNAE, University of Padova, Agripolis, Viale dell'Università, 35020 Legnaro, Padova Italy.
authors.forename: Benedetto
authors.initials: B
authors.lastname: Ruperti
authors.affiliation: Faculty of Biology, Institute of Biology II/Molecular Plant Physiology, University of Freiburg, Freiburg, Germany ; BIOSS Centre for Biological Signaling Studies, University of Freiburg, Freiburg, Germany ; Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, Freiburg, Germany ; Center for Biological Systems Analysis, University of Freiburg, Freiburg, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Palme
keywords: 3D reconstruction; Immunolocalization; Protein–protein interaction; Tissue multi-protein expression; Whole-mount