A safer fluorescent in situ hybridization protocol for cryosections

This paper presents a safer, high-sensitivity fluorescent in situ hybridization (FISH) protocol for cryosections that replaces toxic reagents with low-toxicity alternatives while enabling multiplexed mRNA detection, preserving tissue integrity without proteolytic enzymes, and allowing simultaneous immunostaining.

The Gist

Imagine you are a detective trying to solve a mystery inside a tiny, frozen city (a slice of tissue from a mouse, frog, or fish). Your goal is to find specific "wanted posters" (mRNA molecules) that tell you which genes are active in which cells. This detective work is called Fluorescent In Situ Hybridization (FISH).

For years, to make these posters visible, detectives had to use a very powerful, but dangerous, chemical spray (like formaldehyde or methanol) to "fix" the city in place and make the walls permeable so the posters could be seen. The problem? This spray is toxic, smelly, and bad for the detective's health. It's like trying to solve a crime while wearing a gas mask and risking your lungs every time you open the case file.

The Big Breakthrough:
This paper introduces a safer, cleaner way to do this detective work. The researchers developed a new protocol that swaps the toxic spray for gentle, low-toxicity alternatives (like a special "glyoxal" fixative and ethanol instead of methanol).

Here is how they did it, using some everyday analogies:

1. The "Gentle Hand" Approach (No More Enzymes)

Usually, to see the mRNA posters, scientists have to use "enzymes" (like proteinase K) to chew up the sticky walls of the cells so the probes can get in. Think of this like using a chainsaw to cut a hole in a door just to peek inside. It works, but it often damages the door (the tissue) and requires a lot of fine-tuning.

The New Way: Instead of a chainsaw, this new protocol uses a special "detergent bath" (a mix of Tween 20 and SDS). It's like soaking the door in warm, soapy water. The soap gently loosens the grime, letting the probes slip right through without damaging the door or the furniture inside. This keeps the tissue looking perfect and saves the researchers from handling harsh enzymes.

2. The "Universal Adapter" (Works on Everyone)

The researchers tested this new method on a diverse group of "cities":

• Mouse limbs (mammals)
• Frog and Newt limbs (amphibians that can regenerate)
• Medaka fish eyes (fish)

They found that whether they were looking at a mouse's developing leg or a frog's brain, the new "gentle" fixative worked just as well as the old toxic spray. It was like finding a universal key that opens every door in the city without breaking the lock.

3. The "Double-Exposure" Trick (Seeing Two Things at Once)

One of the coolest features of this new method is that it allows for multimodal detection.

• Old Way: You could see the mRNA (the "wanted poster"), but if you tried to look for the protein (the "criminal" itself) right after, the toxic chemicals often destroyed the evidence.
• New Way: Because the method is so gentle, you can first find the mRNA, and then immediately look for the protein in the same slice of tissue. It's like taking a photo of the suspect's ID card and then, without moving the camera, taking a photo of their face. You get a complete picture of who they are and what they are doing.

4. The "Signal Booster" (HCR and SABER)

To make the "wanted posters" glow brightly enough to see, the researchers used two high-tech amplification systems:

• HCR (Hybridization Chain Reaction): Imagine a line of dominoes. When one falls, it knocks over the next, creating a chain reaction that makes a huge signal.
• SABER: Imagine a tree branch. One probe attaches to the target, and then many smaller "leaves" (fluorescent probes) attach to that branch, making the signal huge and bright.

The paper proves that their new "gentle" protocol works perfectly with both of these amplification systems. They even showed that you can use both systems on the same tissue slice to see different genes at the same time, like watching two different TV channels on the same screen without the picture getting fuzzy.

Why Does This Matter?

• Safety: Researchers no longer need to breathe in toxic fumes or handle dangerous solvents. It's like switching from a gas-powered lawnmower to a quiet, electric one.
• Quality: Because they don't use harsh enzymes or toxic fixatives, the tissue stays in better shape. The "city" looks more natural, making the data more accurate.
• Versatility: It works on mice, frogs, newts, and fish, making it a "one-size-fits-all" tool for biologists studying how animals grow and heal.

In a Nutshell:
This paper is about giving scientists a safer, gentler, and more effective toolkit to map out the genetic activity inside living things. They replaced the "toxic spray" with a "gentle soap," proving that you don't need to be dangerous to get a clear picture of life's smallest details.

Technical Summary

1. Problem Statement

Fluorescent in situ hybridization (FISH) is a critical technique for high-resolution, multiplexed detection of gene transcripts in spatial transcriptomics. However, conventional FISH protocols rely heavily on hazardous reagents that pose significant health risks to researchers:

• Toxic Fixatives: Standard protocols often use formalin (formaldehyde) or glutaraldehyde, which are volatile and carcinogenic.
• Toxic Solvents: Methanol is widely used for delipidation, dehydration, and permeabilization, despite its high toxicity and flammability.
• Enzymatic Damage: To improve probe accessibility, protocols frequently employ proteolytic enzymes (e.g., Proteinase K, pepsin). These can damage tissue morphology and require difficult optimization, potentially degrading target RNA.
• Incompatibility: These harsh conditions often hinder the integration of FISH with immunohistochemistry (IHC), making simultaneous detection of mRNA and proteins difficult.

2. Methodology

The authors developed and validated a "safer" FISH protocol designed to replace toxic reagents with low-toxicity alternatives while maintaining high sensitivity. The core modifications include:

• Alternative Fixatives:
  • Replaced formaldehyde/glutaraldehyde with ALTFiX (a glyoxal-based fixative) and PAXgene® (an ethanol-based fixative).
  • These fixatives allow for direct post-fixation of cryosections on slides without pre-fixation, preserving tissue integrity and compatibility with spatial RNA-seq workflows.
• Solvent Replacement:
  • Replaced methanol with ethanol for all steps involving fixation, delipidation, dehydration, and permeabilization.
• Enzyme-Free Permeabilization:
  • Eliminated proteolytic enzymes (Proteinase K, etc.).
  • Implemented a modified detergent solution containing Tween 20 and Sodium Dodecyl Sulfate (SDS) for a 30-minute room temperature incubation to enhance probe penetration without damaging tissue architecture.
• Workflow Integration:
  • The protocol was adapted for two major signal amplification techniques: in situ Hybridization Chain Reaction (HCR) and Signal Amplification by Exchange Reaction (SABER)-FISH.
  • A "SABER-HCR" workflow was established to perform both amplification methods on the same section for comprehensive multiplexing.
  • The protocol was optimized to allow sequential IHC (specifically for EGFP) immediately after FISH without additional blocking or antigen retrieval steps.

3. Key Contributions

• Safety Enhancement: The protocol successfully eliminates the need for formalin, glutaraldehyde, and methanol, significantly reducing researcher exposure to carcinogens and volatile organic compounds.
• Preservation of Morphology: By avoiding proteolytic digestion, the protocol preserves nuclear architecture and overall tissue integrity better than enzyme-heavy conventional methods.
• Multimodal Compatibility: It enables the seamless integration of mRNA detection (via HCR or SABER) with protein detection (IHC) on the same tissue section.
• Broad Applicability: The method was validated across diverse vertebrate model organisms, including mammals (Mus musculus), amphibians (Xenopus laevis, Pleurodeles waltl), and fish (Oryzias latipes).

4. Results

The authors validated the protocol through extensive experiments:

• Mouse Limb Buds (HCR):
  • Compared ALTFiX fixation against standard 4% PFA.
  • Successfully detected Sox9 and Col2a1 (cartilage markers) with signal patterns and intensities equivalent to PFA.
  • Nuclear architecture (DAPI staining) was well-preserved.
• Frog (Xenopus laevis) Tissues (HCR & HCR-IHC):
  • Detected limb development genes (shh, fgf8, hoxa13, msx1, aggrecan, prg4) in tadpole and adult tissues.
  • HCR-IHC Success: In sox2:egfp knock-in tadpoles, the protocol detected sox2 and shh mRNAs via HCR and subsequently detected EGFP protein via IHC on the same section. This demonstrated that antigenicity is preserved without blocking steps.
  • Wild-type controls confirmed the specificity of the EGFP antibody signal.
• Newt (Pleurodeles waltl) Limb Buds (HCR):
  • Successfully visualized complex expression patterns of limb patterning genes (Fgf8, Fgf10, Hoxa11, Hoxa13, Shh, Hand2, Gli1, Gli3).
  • Achieved clear triple-plex detection (Hand2, Gli1, Gli3) using fluorophores with overlapping spectra (Alexa 488/514/546) without significant bleed-through.
• Medaka (Oryzias latipes) Retina (SABER-FISH):
  • Replaced PFA with ALTFiX in pre-fixed frozen sections.
  • Successfully detected cell-type markers (gja10b for horizontal cells, cabp5a for bipolar cells) with high signal-to-noise ratios, distinguishing specific signals from photoreceptor autofluorescence.
• SABER-FISH in Amphibians and Mice:
  • Validated SABER-FISH detection of Shh in newts, prrx1/sox9 in frogs, and Fgf10/Fgf8 in mice using the safer protocol.
• Integrated SABER-HCR:
  • Demonstrated simultaneous detection of Sox9/Col2a1 (via HCR) and Gdf5 (via SABER) on the same mouse limb section, proving the two amplification systems are compatible and non-interfering.

5. Significance

• Researcher Safety: The protocol offers a practical, high-performance alternative that mitigates health risks associated with handling toxic chemicals in basic biology labs.
• Enhanced Data Quality: By removing enzymatic steps, the protocol reduces tissue damage and variability, leading to more consistent signal detection and better preservation of morphological context.
• Versatility: The ability to combine HCR and SABER-FISH, and to integrate them with IHC, allows for highly complex, multimodal spatial transcriptomics and proteomics studies in a single sample.
• Future-Proofing: The authors note that while formamide (used in hybridization buffers) remains a concern, this protocol lays the groundwork for further safety improvements (e.g., replacing formamide with urea or ethylene carbonate) and potential adaptation to whole-mount or paraffin-embedded samples.

In conclusion, this study provides a robust, safer, and highly versatile workflow for FISH that maintains the sensitivity and multiplexing capabilities of advanced amplification techniques while significantly improving laboratory safety and tissue preservation.