PMID- 37993896
OWN - NLM
STAT- PubMed-not-MEDLINE
LR  - 20231125
IS  - 1746-4811 (Print)
IS  - 1746-4811 (Electronic)
IS  - 1746-4811 (Linking)
VI  - 19
IP  - 1
DP  - 2023 Nov 22
TI  - A rapid and sensitive, multiplex, whole mount RNA fluorescence in situ 
      hybridization and immunohistochemistry protocol.
PG  - 131
LID - 10.1186/s13007-023-01108-9 [doi]
LID - 131
AB  - BACKGROUND: In the past few years, there has been an explosion in single-cell 
      transcriptomics datasets, yet in vivo confirmation of these datasets is hampered 
      in plants due to lack of robust validation methods. Likewise, modeling of plant 
      development is hampered by paucity of spatial gene expression data. RNA 
      fluorescence in situ hybridization (FISH) enables investigation of gene 
      expression in the context of tissue type. Despite development of FISH methods for 
      plants, easy and reliable whole mount FISH protocols have not yet been reported. 
      RESULTS: We adapt a 3-day whole mount RNA-FISH method for plant species based on 
      a combination of prior protocols that employs hybridization chain reaction (HCR), 
      which amplifies the probe signal in an antibody-free manner. Our whole mount HCR 
      RNA-FISH method shows expected spatial signals with low background for gene 
      transcripts with known spatial expression patterns in Arabidopsis inflorescences 
      and monocot roots. It allows simultaneous detection of three transcripts in 3D. 
      We also show that HCR RNA-FISH can be combined with endogenous fluorescent 
      protein detection and with our improved immunohistochemistry (IHC) protocol. 
      CONCLUSIONS: The whole mount HCR RNA-FISH and IHC methods allow easy 
      investigation of 3D spatial gene expression patterns in entire plant tissues.
CI  - (c) 2023. The Author(s).
FAU - Huang, Tian
AU  - Huang T
AD  - Department of Biology, University of Pennsylvania, Philadelphia, PA, 19104, USA.
FAU - Guillotin, Bruno
AU  - Guillotin B
AD  - Department of Biology, Center for Genomics and Systems Biology, New York 
      University, New York, NY, 10003, USA.
FAU - Rahni, Ramin
AU  - Rahni R
AD  - Department of Biology, Center for Genomics and Systems Biology, New York 
      University, New York, NY, 10003, USA.
FAU - Birnbaum, Kenneth D
AU  - Birnbaum KD
AD  - Department of Biology, Center for Genomics and Systems Biology, New York 
      University, New York, NY, 10003, USA.
FAU - Wagner, Doris
AU  - Wagner D
AD  - Department of Biology, University of Pennsylvania, Philadelphia, PA, 19104, USA. 
      wagnerdo@sas.upenn.edu.
LA  - eng
GR  - LT000972/2018-L/Human Frontiers of Science/
GR  - 2319036/Division of Integrative Organismal Systems/
PT  - Journal Article
DEP - 20231122
PL  - England
TA  - Plant Methods
JT  - Plant methods
JID - 101245798
PMC - PMC10666358
OTO - NOTNLM
OT  - Fluorescent protein
OT  - Hybridization chain reaction
OT  - Immunohistochemistry
OT  - RNA-FISH
OT  - Whole mount
COIS- The authors declare that they have no competing interests.
EDAT- 2023/11/23 06:42
MHDA- 2023/11/23 06:43
PMCR- 2023/11/22
CRDT- 2023/11/23 00:05
PHST- 2023/05/29 00:00 [received]
PHST- 2023/11/10 00:00 [accepted]
PHST- 2023/11/23 06:43 [medline]
PHST- 2023/11/23 06:42 [pubmed]
PHST- 2023/11/23 00:05 [entrez]
PHST- 2023/11/22 00:00 [pmc-release]
AID - 10.1186/s13007-023-01108-9 [pii]
AID - 1108 [pii]
AID - 10.1186/s13007-023-01108-9 [doi]
PST - epublish
SO  - Plant Methods. 2023 Nov 22;19(1):131. doi: 10.1186/s13007-023-01108-9.