In situ hybridization histochemistry: a new method for processing material stored for several years

Area of Science:

• Neuroscience
• Molecular Biology
• Biochemistry

Background:

• Detecting specific messenger RNAs (mRNAs) in brain tissue is crucial for understanding neurological function and disease.
• Traditional methods for in situ hybridization require fresh tissue or specific preservation techniques, limiting long-term storage and accessibility.
• RNase activity and sample degradation are significant challenges in preserving RNA integrity in stored tissue sections.

Purpose of the Study:

• To develop and validate a novel in situ hybridization protocol for detecting specific mRNAs in cryoprotected brain tissue sections stored for extended periods.
• To assess the efficacy and specificity of the protocol in rat, monkey, and human brain tissues.
• To compare the results with traditional methods using fresh or immediately processed tissue.

Main Methods:

• Brain tissues from rats, monkeys, and humans were sectioned and stored in an ethylene glycol-based cryoprotectant solution at -20°C for up to several years.
• Oligonucleotide probes targeting specific rat and human mRNAs (preproenkephalin, tyrosine hydroxylase) were synthesized and labeled.
• In situ hybridization was performed in sterile culture dishes to minimize RNase contamination, with specific probe volumes optimized for each species.

Main Results:

• The developed protocol successfully detected specific mRNAs in rat, monkey, and human brain sections stored for extended periods (up to 5 years for monkeys, 1 year for humans).
• Rat brain sections processed with this protocol showed higher specificity and reduced non-specific background compared to control sections mounted immediately.
• Excellent resolution was achieved for mRNA detection in monkey and human brain sections after long-term cryoprotectant storage.

Conclusions:

• This protocol enables reliable detection of specific mRNAs in long-term stored, cryoprotected brain tissue, overcoming RNase degradation issues.
• The method offers improved specificity and reduced background, enhancing the accuracy of in situ hybridization studies.
• This technique expands the utility of archived tissue samples for molecular neuroscience research.