1：Experimental Design and Method Selection:

The study designed G-FISH using graphene oxide (GO) nanosheets to quench fluorophore-labeled peptide nucleic acid (PNA) probes, with hybridization to target RNAs in tissues for fluorescence recovery. Theoretical models include π-π interactions for probe attachment and hybridization kinetics.

2：Sample Selection and Data Sources:

Samples included normal mouse brains, primary cultured hippocampal neurons, Alzheimer's disease model mouse brains (5 × FAD mice), and human glioblastoma multiforme (GBM) tumor tissues. Tissues were formalin-fixed paraffin-embedded (FFPE), frozen, or CLARITY-transparent.

3：List of Experimental Equipment and Materials:

Equipment includes microtome for sectioning, confocal microscope (Zeiss LSM 510 META), GloMax Discover system for fluorescence measurement, ABI 7500 instrument for RT-PCR. Materials include GO nanosheets (Lemonex Corporation), PNA oligomers (Panagene), fluorophores (FAM, TAMRA), antibodies (e.g., GFAP), and various reagents for tissue processing and staining.

4：Experimental Procedures and Operational Workflow:

Tissues were deparaffinized, rehydrated, and treated with GO-PNA complexes. Incubation was at room temperature for up to 16 hours. For CLARITY, brains were cleared using hydrogel and electrophoresis. Fluorescence imaging and RT-PCR were performed for validation.

5：Data Analysis Methods:

Fluorescence intensities were quantified using imaging software, and statistical analysis (Student's t-test) was applied to compare groups. Correlation between G-FISH and RT-PCR data was assessed.