[Histonet] CISH advice for zoox in FFPE Exaiptasia

[Tilley, Julie]

Hello everyone, we are looking for some advice.
We're trying to localize zooxanthellae in Formalin-fixed paraffin-embedded Exaiptasia tissues using chromogenic in situ hybridization, but so far we haven't had any success. We've tested multiple retrieval conditions - heat retrieval at pH 6 and pH 8, proteinase K (25, 50, and 100 µg/mL), cellulase (1 mg/mL), and HCl (0.2N) - along with several DIG labeled probes targeting Symbiodinium clade B genes (published 18S "SymB_DIG" by Yokouchi et al. 2003, and designed in house 28S "B1-rev_DIG", ITS2, cytochrome b "cob-rev_DIG", and chloroplast "cp23S-rev_DIG").
qPCR shows strong amplification (Cq < 25) for all these targets, so we're confident the sequences are present in the tissues. We also included positive controls in every experiment, and those consistently worked as expected.
If anyone has suggestions on what we should try next, we'd really appreciate your insight.
Thanks so much for any guidance you can share!

Probe sequences:
ITS2-rev_DIG: 5'-/5DigN/GCAAACAAGCCATTTACGAAC -3'
cob-rev_DIG: 5'-/5DigN/ATCTAGAGCTTACCCACACG -3'
cp23S-rev_DIG: 5'-/5DigN/CCTTTAGTGTCAGCAGCATA -3'
SymB_DIG: 5'-/5DigN/GGGCAAGATCGAAAACTTG-3'
B1-rev_DIG: 5'-/5DigN/CTTGTTTGCTATTGGTCTCG-3'

References:
Yokouchi, H., Takeyama, H., Miyashita, H., Maruyama, T., & Matsunaga, T. (2003). In situ identification of symbiotic dinoflagellates, the genus Symbiodinium with fluorescence-labeled rRNA-targeted oligonucleotide probes. Journal of Microbiological Methods, 53(3), 327-334. https://doi.org/10.1016/s0167-7012(02)00250-6.

Protocol from: Conway, C. M., Purcell, M. K., Elliott, D. G., & Hershberger, P. K. (2014). Detection of Ichthyophonus by chromogenic in situ hybridization. Journal of Fish Diseases, 38(9), 853-857. https://doi.org/10.1111/jfd.12300




Julie Tilley
Biological Science Lab Tech
USGS-National Wildlife Health Center
820 Mililani Street, Suite 300
Honolulu, HI 96813
(O): 808-535-9112
(C): 808-970-0355