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Journal of Integrative Agriculture  2023, Vol. 22 Issue (2): 464-470    DOI: 10.1016/j.jia.2022.08.038
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An optimized protocol using Steedman’s wax for high-sensitivity RNA in situ hybridization in shoot apical meristems and flower buds of cucumber

WANG Cui1*, SUN Jin-jing2*, YANG Xue-yong2, WAN Li2, ZHANG Zhong-hua1, ZHANG Hui-min1

1 College of Horticulture, Qingdao Agricultural University/Engineering Laboratory of Genetic Improvement of Horticultural Crops of Shandong Province/Laboratory of Quality & Safety Risk Assessment for Fruit (Qingdao), Ministry of Agriculture and Rural Affairs, Qingdao 266109, P.R.China

2 Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences/Key Laboratory of Biology and Genetic Improvement of Horticultural Crops of the Ministry of Agriculture and Rural Affairs/Sino-Dutch Joint Laboratory of Horticultural Genomics, Beijing 100081, P.R.China.

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In situ mRNA hybridization (ISH) is a powerful tool for examining the spatiotemporal expression of genes in shoot apical meristems and flower buds of cucumber.  The most common ISH protocol uses paraffin wax; however, embedding tissue in paraffin wax can take a long time and might result in RNA degradation and decreased signals.  Here, we developed an optimized protocol to simplify the process and improve RNA sensitivity.  We combined embedding tissue in low melting-point Steedman’s wax with processing tissue sections in solution, as in the whole-mount ISH method in the optimized protocol. Using the optimized protocol, we examined the expression patterns of the CLAVATA3 (CLV3) and WUSCHEL (WUS) genes in shoot apical meristems and floral meristems of Cucumis sativus (cucumber) and Arabidopsis thaliana (Arabidopsis).  The optimized protocol saved 4–5 days of experimental period compared with the standard ISH protocol using paraffin wax.  Moreover, the optimized protocol achieved high signal sensitivity.  The optimized protocol was successful for both cucumber and Arabidopsis, which indicates it might have general applicability to most plants

Keywords:  cucumber        in situ hybridization        Steedman’s wax        paraffin wax  
Received: 23 March 2022   Accepted: 09 May 2022
Fund: We thank Dr. Hu Bowen (Hunan Agricultural University, China) for valuable suggestions on this program.  This work was supported by the National Natural Science Foundation of China (32002036).

About author:  WANG Cui, E-mail:; SUN Jin-jing, E-mail:; Correspondence ZHANG Hui-min, Tel/Fax: +86-532-58957760, E-mail: * These authors contributed equally to this study.

Cite this article: 

WANG Cui, SUN Jin-jing, YANG Xue-yong, WAN Li, ZHANG Zhong-hua, ZHANG Hui-min. 2023. An optimized protocol using Steedman’s wax for high-sensitivity RNA in situ hybridization in shoot apical meristems and flower buds of cucumber. Journal of Integrative Agriculture, 22(2): 464-470.

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