Fluorescence In Situ Hybridization (FISH) is a molecular technique used to identify the presence and spatial distribution of specific RNA transcripts within cells.

Chromosomal abnormality test: In prenatal diagnosis, FISH can be used to detect Down syndrome.

Cancer marker test: FISH can detect Philadelphia chromosome (Ph chromosome) translocations common in patients with chronic myeloid leukemia (CML), where chromosome 22 is slightly shorter than that of normal people and chromosome 9 is slightly longer.

Fluorescence in situ hybridization technique uses a known fluorescently labeled single-stranded DNA/RNA as a probe, based on the principle of base complementary pairing, through steps such as denaturation, annealing and renaturation, specifically binds to the target single-stranded DNA/RNA in the sample to be tested to form detectable hybrid double-stranded DNA/RNA. By observing and counting the fluorescence signal with a fluorescence microscope or laser confocal microscope, the morphology and distribution of the cells or organelles stained after hybridization with the specific probe can be determined, or the location of the DNA region bound to the fluorescent probe and RNA molecules in chromosomes and other organelles can be identified (Figure 1).

• High sensitivity and specificity: It can precisely detect and locate target nucleic acid sequences, and effectively distinguish target sequences from non-target sequences even in complex cellular environments.
• Support for multi-color fluorescence labeling: Capable of detecting multiple target sequences simultaneously, significantly enhancing detection efficiency and accuracy.
• Intuitive visualization: With the aid of a fluorescence microscope, the distribution and localization of target sequences within cells or tissues can be directly observed.
• Wide range of application: Applicable to both dividing and non-dividing cells, capable of detecting recessive gene deletions, gene translocations and low-level gene chimeras, and operable on paraffin-embedded tissue (PET) sections.

The core steps of a FISH experiment are: sample preparation (fixing cells or tissues on slides) → pretreatment (fixing exposed targets through digestion, deproteinization and dehydration) → hybridization (binding probes to target sequences) → washing (removing probes that do not specifically bind) → counterstaining and mounting (enhancing signals and providing protection) → fluorescence microscopy observation and analysis.

The key to sample pretreatment is to expose the nucleic acid within the cells or tissues to ensure effective binding of the probe, a process that typically involves protease digestion and gradient ethanol dehydration operations. The tissue pretreatment steps are shown in Figure 2.

After the tissue pretreatment was completed, the tissue on the slide was enveloped (circled) using a hydrophobic barrier pen and then incubated at room temperature in a protease solution. After washing, transfer the tissue to a incubator for hybridization for 2 hours before proceeding with the amplification steps in sequence. After FISH probe treatment, the tissue should be washed first and then sealed with complete horse serum. Incubation of the primary antibody is carried out overnight at 4°C to ensure full binding of the antibody to the antigen; Secondary antibody incubation was carried out at room temperature for 2 hours (Figure 3).

Note: The design of the probe should be highly specific to ensure that it hybridizes only with the target sequence and avoids non-specific binding with other sequences. The hybridization temperature is typically 37°C or 42°C and the reaction time is generally 16-20 hours; However, the duration of the hybridization reaction is related to the probe length and cell permeability. If the cells have completed sufficient permeability treatment, the oligonucleotide probe only takes 2 to 6 hours to complete the hybridization. In addition, the purpose of the washing step is to remove unbound probes in order to reduce non-specific signal interference. This step typically uses a buffer with an appropriate salt concentration. During the operation, it is necessary to keep the environmental temperature above 20℃ and multiple washes should be carried out. The washing after hybridization should follow a general principle: the salt solution concentration should be adjusted in a gradient from high to low, while the temperature should be gradually increased in a sequence from low to high.

The combination of mRNA FISH and IHC imaging can be used to characterize the efficacy of complement-targeted therapies in AD models, or to study gene expression in combination with other protein aggregates associated with the pathology of Alzheimer's disease, such as tau and synuclein.

"Example": Combining FISH and IHC to detect glial complement expression in amyloid plaques in mouse brains

• Use mRNA FISH to detect astrocyte and microglia-specific complement expression
• Signal amplification for detecting Aβ plaques in a TauPS2APP mouse model
• The relationship between glial complement expression and Aβ plaques was measured using mRNA FISH
• Complete the full-film imaging and spatial gene expression analysis workflow within one week

Complement proteins promote neurodegenerative changes in Alzheimer's disease (AD), which are secreted by glial cells that surround beta-amyloid plaques. For this purpose, the authors proposed an optimized Aβ plaque detection scheme based on Tyramide-digoxigenin signaling amplification technology (as shown in Figure 4). Combined with multiplex mRNA in situ fluorescence hybridization (FISH) for detecting glial cell-specific complement expression in the proximal region of Aβ plaques in the TauPS2APP mouse model. The results showed that higher signal-to-noise ratios were achieved in consecutive FISH-IHC experiments through signal amplification processing, and sensitive detection of diffuse Aβ plaques was realized (as shown in Figure 5).

FISH-Flow technology enables quantitative fluorescence analysis at the single-cell level by hybridizing fluorescent oligonucleotides with RNA using flow cytometry.

"Example": FISH-Flow for quantifying nascent and mature ribosomal RNA in mouse and human cells.

Researchers have developed a FISH-Flow method for quantifying nascent 47S rRNA as well as mature 18S and 28S rRNA in mouse and human cells, which can also be combined with DNA staining techniques for rRNA quantification across cell cycle stages (as shown in Figure 6).

Both nascent and mature ribosomal RNA are abundant in cells. Therefore, as shown in Figure 7A, the FISH probe targeting rRNA has a high signal-to-noise ratio. Staining with DNA-binding DAPI dyes divides the cell population into G1/S/G2-M phases and quantifies rRNA at each stage of the cell cycle, as shown in Figure 7B.

Sequential fluorescence in situ hybridization (seqFISH) is an advanced biomolecular analysis method that combines imaging techniques with combinatorial molecular barcoding techniques using probes with known sequences, For multi-round hybridization imaging of whole RNA within cells/tissues, the gene ID is determined by analyzing the combination of fluorescence signals in the barcode region of the probe's two arms, and images are generated by associating specific position information with the gene ID. The seqFISH method was able to obtain high-resolution information on cell type, state, and neighborhood relationship in the native tissue and organ environment (Figure 8).

A team led by Nobel Prize winner in Chemistry and CRISPR gene editing technology pioneer Jennifer Doudna published in Nature Biotechnology an article titled "Single-molecule live cell RNA imaging with Crispr-csm". The team developed a Single-molecule live cell fluorescence in situ hybridization (smLiveFISH) technique.

smLiveFISH: The technique uses RNA from Streptococcus thermophilus to target the type III-A CRISPR-Csm system and multiplex guide RNA, enabling researchers to visualize individual RNA molecules directly and efficiently in a variety of cell types, including primary cells, and track the dynamic trajectories of individual mRNA molecules in different types of living cells (Figure 9).

As shown in the figure, GFP-labeled Csm complexes label individual NOTCH2 mRNAs (left in Figure 10 a and b), smFISH probes label NOTCH2 mRNAs (middle in Figure 10 a and b), and superposition images of the two show co-localization (right in Figure 10 a and b). The proportion of co-localization between Csm complex lesions and smFISH lesions, as well as the proportion of transfected cells with Csm complex-labeled lesions, can be quantified (Figure 10c-d).

Verified by two-color imaging, the Csm complex successfully labeled endogenous NOTCH2 mRNA and achieved robust detection in multiple cell types without affecting mRNA stability, decay rate, localization, and protein expression. In addition, the smLiveFISH technique also successfully labeled MAP1B mRNA. The accuracy of the signal was verified through co-localization experiments, and the spatial distribution characteristics of its enrichment in the periphery of the cell were revealed without interfering with mRNA stability, decay rate, localization, and protein expression. Further evidence that the technique is applicable to the visualization study of different RNA molecules.

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