ISHpalette™ Short hairpin amplifier

ISHpalette™ Short hairpin amplifier is a fluorescent hairpin DNA reagent developed as an enhanced version of the traditional in situ HCR method for detecting target molecules in tissue sections.

The in situ Hybridization Chain Reaction (HCR) method utilizes the self-catalyzed elongation of nucleic acids with a hairpin structure 1) to initiate a chain polymerization reaction on labeled DNA within tissue sections, thereby enabling visualization of target nucleic acids 2).

The hybridization of two probes with sequences that initiate the HCR (polymerization reaction) of the hairpin DNA, followed by the subsequent HCR, results in fluorescently labeled DNA specifically binding to the target mRNA.

HCR occurs only when the two probes hybridize in the correct positions, minimizing nonspecific reactions. The ISHpalette™ is a further shortened version of the hairpin DNA, eliminating the need for the conventional permeation treatment 3). Furthermore, because the hybridization time per molecule is reduced, the reaction reaches plateau faster, resulting in higher signal intensity.

1) Dirks and Pierce 2004 PNAS
2) Choi et al. 2010 Nat Biotec; 2014 ACS nano; 2018 Development
3) Tsuneoka and Funato 2020 Front. Mol. Neurosci.

Modified in situ Hybridization Chain Reaction Using Short Hairpin DNAs.
Tsuneoka Y et al.(2020) Front. Mol. Neurosci.

The mRNA of Vglut2 (green), Sik3 (red), and Vgat (magenta) were detected on mouse brain sections. Each mRNA molecule can be detected at a level where it appears as a single granule.

Flowchart: in situ HCR with ISHpalette™ for Frozen Sections

No special equipment or reagents required.
Permeabilization treatments—such as Proteinase K, acetylation, high-temperature treatment, or RNase treatment—are not necessary, resulting in a simplified protocol and faster staining process.

A comparison of staining performance was conducted between fluorescently labeled hairpin DNA purified by a general oligo manufacturer (standard PAGE purification) and our proprietary PAGE-purified product. Our original product, developed in collaboration with Toho University, demonstrated higher fluorescent labeling efficiency and improved reactivity, resulting in stronger fluorescence signals. The comparison was performed using mouse liver tissue targeting Albumin, Cyp2e1, Glul.

The mRNA of Penk (top), Vglut2 (middle), and Nts (bottom) in mouse brain sections was detected using both the conventional HCR method and ISHpalette™. Signal intensity was compared between the two approaches. The results clearly show that ISHpalette™—our shortened hairpin DNA—provides improved tissue permeation and reactivity. Both methods were conducted without Proteinase K (ProK) treatment.

→Here is the sequence information for each hairpin DNA (Excel: 11KB).

We accept custom orders for combinations of hairpin DNA and labels that differ from our standard products, custom-made hairpin DNA (refer to the species compatibility table below), and other labeled products (such as biotin, ATTO647N, etc.).

SaraFluor™ 488: A dye manufactured by a Japanese manufacturer, known for its resistance to photobleaching. Here is the page for SaraFluor™ 488.

Note: Studies on other species are currently ongoing. You can proceed with using the product after confirming through negative controls that there are no non-specific reactions.
For custom-made hairpin DNA (S10, A111, A127), please contact us.

→Here is the assessment protocol for evaluating hairpin DNA usability.

We offer custom probe design services. Based on the target sequence, we select the optimal probe sequences designed with consideration of factors such as Tm values and GC content. The final deliverable includes the probe sequence information along with the binding sequence for the hairpin DNA.

Note:
Using the provided sequence information, you will need to order the oligonucleotides from a synthesis company on your own.
If you wish to design the probe yourself, please refer to the user manual.

→An example of a designed probe (positive control: Gapdh) is available here (Excel: 10KB).

For each region, two types of target probes are designed, one for the 5′ side and one for the 3′ side. Detection cannot be achieved with only one type of hybridization, so you can use a probe with only one of the target probes for the 5′ or 3′ end as a negative control.

Note:

• This product is for research use only and cannot be used for clinical applications.
• ISHpalette™ is developed and commercialized in collaboration with Associate Professor Hiroyuki Tsuneoka from the Department of Anatomy, School of Medicine, Toho University, using the technology patented under Patent No. 7482506.
• SaraFluor™ is a registered trademark of Goryo Chemical, Co., Ltd.