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Protocol: an improved and universal procedure for whole-mount immunolocalization in plants.

Pasternak T, Tietz O, Rapp K, Begheldo M, Nitschke R, Ruperti B, Palme K - Plant Methods (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Biology, Institute of Biology II/Molecular Plant Physiology, University of Freiburg, Freiburg, Germany.

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.

No MeSH data available.


Related in: MedlinePlus

Protein immunolocalization in different Triticum aestivum organs. Three days old wheat seedlings were fixed for 30 min in formaldehyde. Anti-PIN1 mouse monoclonal primary antibody (clone 10A7) diluted 1:50 and Alexa Fluor® 488 goat anti-mouse IgG as secondary antibody diluted 1:800 were used (shown in green color) (a–e); anti-PIN2 Guinea pig primary antibody plus Goat anti-Guinea pig IgG Alexa Fluor® 647 conjugate as secondary antibody diluted 1:800 (shown in red color) (e) and anti-BIP2 (AS09 615) rabbit primary antibody plus Goat anti-rabbit IgG DyLight® 549 conjugate (AS09 642) as secondary antibody diluted 1:3000 (shown in red color) (f) were used. Co-staining with DAPI visualizes nuclei (blue). a leaf; b meristem; c coleoptile; d–f roots. Arrows point polarly located PIN1 and PIN2 proteins. Scale bar 20 µm
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Fig6: Protein immunolocalization in different Triticum aestivum organs. Three days old wheat seedlings were fixed for 30 min in formaldehyde. Anti-PIN1 mouse monoclonal primary antibody (clone 10A7) diluted 1:50 and Alexa Fluor® 488 goat anti-mouse IgG as secondary antibody diluted 1:800 were used (shown in green color) (a–e); anti-PIN2 Guinea pig primary antibody plus Goat anti-Guinea pig IgG Alexa Fluor® 647 conjugate as secondary antibody diluted 1:800 (shown in red color) (e) and anti-BIP2 (AS09 615) rabbit primary antibody plus Goat anti-rabbit IgG DyLight® 549 conjugate (AS09 642) as secondary antibody diluted 1:3000 (shown in red color) (f) were used. Co-staining with DAPI visualizes nuclei (blue). a leaf; b meristem; c coleoptile; d–f roots. Arrows point polarly located PIN1 and PIN2 proteins. Scale bar 20 µm

Mentions: Previously published immunolocalization protocols [4] require at least two working days and cannot be applied to non-transparent tissues. These protocols have been applied for analysis of the root meristem of Arabidopsis thaliana, while for other plant species and for more dense tissues of Arabidopsis (e.g. hypocotyls or leaves) researchers prefer to use paraplast sections which are labor and time consuming and do not allow 3D reconstruction. For example, Bustos-Sanmamed et al. [2] suggested to use paraplast sections for immunolocalization in Medicago plants, which are extremely time and labor consuming. In our hands Medicago can be subjected to whole-mount immunolocalization in any organ with further 3D reconstruction. Our whole-mount protocol is applicable to the analysis of any plant species and organ including non-transparent tissues. It is also easily applicable to suspension cultures and can be completed for most specimens in 5–6 h. Detection of proteins deep inside tissues requires a fine balance between fixation, clearing of tissues, cell wall digestion and permeabilisation. Through improved tissue clearing combined with tissue-specific combinations of cell wall degrading enzymes, proteins can be detected e.g. in ovules of intact pistils or xylem-parenchyma cells of hypocotyls while keeping the outer cell structures intact (Figs. 2, 3). The excellent tissue preservation is demonstrated by labeling of microtubules and actin in the elongation zone of Arabidopsis roots (Fig. 4), which often appeared destroyed using previously published automated whole-mount method [3]. Due to the use of small volumes in Microarray slides, the procedure described here reduces the amount of reagents and limits the use of particularly precious antibodies, but also allows handling of specimens up to 1 cm wide. The general applicability of the protocol was successfully tested for localization of PIN proteins in root and flower tissues from Medicago sativa, Triticum aestivum, Lycopersium esculentum, and Hedera helix (Figs. 5, 6, 7). The fixation procedure using ethyldimethylaminopropyl carbodiimide (EDAC, carboxyl activating agent for hormones bonding with proteins) and formaldehyde was optimized for detection of low molecular weight molecules (e.g. auxin) with antibodies (Fig. 8).Fig. 2


Protocol: an improved and universal procedure for whole-mount immunolocalization in plants.

Pasternak T, Tietz O, Rapp K, Begheldo M, Nitschke R, Ruperti B, Palme K - Plant Methods (2015)

Protein immunolocalization in different Triticum aestivum organs. Three days old wheat seedlings were fixed for 30 min in formaldehyde. Anti-PIN1 mouse monoclonal primary antibody (clone 10A7) diluted 1:50 and Alexa Fluor® 488 goat anti-mouse IgG as secondary antibody diluted 1:800 were used (shown in green color) (a–e); anti-PIN2 Guinea pig primary antibody plus Goat anti-Guinea pig IgG Alexa Fluor® 647 conjugate as secondary antibody diluted 1:800 (shown in red color) (e) and anti-BIP2 (AS09 615) rabbit primary antibody plus Goat anti-rabbit IgG DyLight® 549 conjugate (AS09 642) as secondary antibody diluted 1:3000 (shown in red color) (f) were used. Co-staining with DAPI visualizes nuclei (blue). a leaf; b meristem; c coleoptile; d–f roots. Arrows point polarly located PIN1 and PIN2 proteins. Scale bar 20 µm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4625903&req=5

Fig6: Protein immunolocalization in different Triticum aestivum organs. Three days old wheat seedlings were fixed for 30 min in formaldehyde. Anti-PIN1 mouse monoclonal primary antibody (clone 10A7) diluted 1:50 and Alexa Fluor® 488 goat anti-mouse IgG as secondary antibody diluted 1:800 were used (shown in green color) (a–e); anti-PIN2 Guinea pig primary antibody plus Goat anti-Guinea pig IgG Alexa Fluor® 647 conjugate as secondary antibody diluted 1:800 (shown in red color) (e) and anti-BIP2 (AS09 615) rabbit primary antibody plus Goat anti-rabbit IgG DyLight® 549 conjugate (AS09 642) as secondary antibody diluted 1:3000 (shown in red color) (f) were used. Co-staining with DAPI visualizes nuclei (blue). a leaf; b meristem; c coleoptile; d–f roots. Arrows point polarly located PIN1 and PIN2 proteins. Scale bar 20 µm
Mentions: Previously published immunolocalization protocols [4] require at least two working days and cannot be applied to non-transparent tissues. These protocols have been applied for analysis of the root meristem of Arabidopsis thaliana, while for other plant species and for more dense tissues of Arabidopsis (e.g. hypocotyls or leaves) researchers prefer to use paraplast sections which are labor and time consuming and do not allow 3D reconstruction. For example, Bustos-Sanmamed et al. [2] suggested to use paraplast sections for immunolocalization in Medicago plants, which are extremely time and labor consuming. In our hands Medicago can be subjected to whole-mount immunolocalization in any organ with further 3D reconstruction. Our whole-mount protocol is applicable to the analysis of any plant species and organ including non-transparent tissues. It is also easily applicable to suspension cultures and can be completed for most specimens in 5–6 h. Detection of proteins deep inside tissues requires a fine balance between fixation, clearing of tissues, cell wall digestion and permeabilisation. Through improved tissue clearing combined with tissue-specific combinations of cell wall degrading enzymes, proteins can be detected e.g. in ovules of intact pistils or xylem-parenchyma cells of hypocotyls while keeping the outer cell structures intact (Figs. 2, 3). The excellent tissue preservation is demonstrated by labeling of microtubules and actin in the elongation zone of Arabidopsis roots (Fig. 4), which often appeared destroyed using previously published automated whole-mount method [3]. Due to the use of small volumes in Microarray slides, the procedure described here reduces the amount of reagents and limits the use of particularly precious antibodies, but also allows handling of specimens up to 1 cm wide. The general applicability of the protocol was successfully tested for localization of PIN proteins in root and flower tissues from Medicago sativa, Triticum aestivum, Lycopersium esculentum, and Hedera helix (Figs. 5, 6, 7). The fixation procedure using ethyldimethylaminopropyl carbodiimide (EDAC, carboxyl activating agent for hormones bonding with proteins) and formaldehyde was optimized for detection of low molecular weight molecules (e.g. auxin) with antibodies (Fig. 8).Fig. 2

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Biology, Institute of Biology II/Molecular Plant Physiology, University of Freiburg, Freiburg, Germany.

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.

No MeSH data available.


Related in: MedlinePlus