A Hemisphere-Separated Protocol for Morphological Observation and Combined RNA-Protein Extraction in Murine Brain Regions

Summary

This protocol bisects murine brains. One hemisphere is used for morphological staining, and the other for RNA and protein extraction. The strategy decreases animal consumption, follows the 3Rs principles and minimizes inter-animal variation.

Abstract

In daily neuroscience research, morphological staining and nucleic acid/protein molecular assays typically require separate experimental animals, which increases animal use and is inconsistent with the 3Rs animal welfare principle. This protocol establishes a tissue allocation workflow for murine brain tissue that enables paired morphological and molecular analyses from a single animal. After transcardial perfusion with 0.9% saline, the brain is bisected into two hemispheres. One hemisphere is fixed in 4% paraformaldehyde for immunohistochemistry and immunofluorescence; sections stored in a sucrose-ethylene glycol-polyvinylpyrrolidone (PVP) solution maintain reliable staining quality for up to seven years. The contralateral hemisphere is cryopreserved at -80 °C for combined RNA and protein extraction, with yields sufficient for downstream qPCR and Western Blot. Tested on 9–10 mg entorhinal cortex punches, this method produces lower RNA and protein yields than separate extraction of RNA or protein individually, yet the recovered biomolecules remain adequate for molecular detection. This workflow lowers animal consumption and removes inter-individual differences between paired morphological and molecular analyses. It provides a feasible tissue preparation method for multidimensional profiling of discrete murine brain subregions.

Introduction

Neuroscience research has expanded dramatically over recent decades, uncovering key mechanisms governing human brain physiology, neurological disease onset, and therapeutic development. Animal models remain indispensable for neuroscientific inquiry, providing controllable, reproducible platforms to explore neural development, physiological function, and pathological processes. Experimental manipulation and histological observation in murine subjects allow researchers to characterize neuronal signaling, neural circuit formation, and neural-behavioral associations. Parallel to technical progress, animal experimental ethics has become a central concern. The 3Rs principles (Replacement, Reduction, and Refinement) are now globally accepted standards for all animal studies^1,2, requiring researchers to minimize animal use while maintaining reliable experimental outcomes.

Innovative tools such as conditional knockout systems permit precise genetic manipulation within discrete subregions of the murine brain³. These advances have fueled intensive research into microscale brain compartments, such as hippocampal CA1, CA3, and DG, that regulate learning, memory, and mood⁴. Decoding the high-order functions of these tiny brain regions demands coordinated morphological, transcriptional, and protein-level measurements. Unfortunately, their scarce tissue volume creates major technical obstacles for parallel multi-index detection.

Morphological staining, RNA quantification, and protein immunodetection are three routine core readouts in neuroscience. Traditional research designs require three separate cohorts of murine animals to complete these three types of testing. This practice drastically increases animal expenditure and introduces considerable inter-animal biological variation, which weakens comparability between histological and molecular datasets. A unified workflow that acquires matched morphological and molecular signals from one single animal would greatly address these two critical limitations and better comply with the Reduction principle of the 3Rs.

Here, we report a reproducible hemispheric separation protocol for murine brain tissue. After systemic saline perfusion, the whole brain is divided into two hemispheres: one hemisphere is fixed for morphological examinations, and the contralateral hemisphere is cryopreserved for simultaneous RNA and protein co-extraction. Tissues fixed and stored in sucrose-ethylene glycol-polyvinylpyrrolidone (PVP) protective buffer maintain stable, high-quality staining signals for immunohistochemistry and immunofluorescence for up to seven years. We validated this method using tiny 9–10 mg punches of the entorhinal cortex, tissue lighter than commonly investigated regions such as the hippocampus and prefrontal cortex. The successful detection of this minute tissue sample indicates that smaller brain subregions such as the hypothalamus and amygdala are also compatible with this workflow. Although the protocol generates lower RNA and protein concentrations than specialized single-purpose extraction kits, the isolated biomolecules possess adequate quality to support standard downstream qPCR and Western blot analysis. This workflow lowers animal consumption and removes inter-individual differences between paired morphological and molecular analyses. It provides a feasible tissue preparation method for multidimensional profiling of discrete murine brain subregions.

Protocol

Ethics statement

All animal experiments performed in this study were approved by the Institutional Animal Care and Use Committee of Dalian Medical University (Approval No. AEE18026 for rat experiments and No. AEE24261 for mouse experiments), People’s Republic of China. All experimental protocols were implemented in accordance with the ethical guidelines for laboratory animals and fully complied with the 3Rs principles (Reduction, Replacement, and Refinement) for animal experimentation. Throughout the entire experimental process, rigorous measures were adopted to effectively alleviate animal suffering and distress. Meanwhile, the number of experimental animals was limited to the minimum quantity required to ensure the reliability and validity of statistical analysis results. See the Table of Materials for a complete list of reagents, consumables, and equipment used in this protocol.

1. Experimental animal preparation and transcardial perfusion surgery

1. Anesthesia and pre-operative preparation
   1. Use specific-pathogen-free (SPF) grade adult male Sprague-Dawley rats or C57BL/6 mice for this protocol. House the animals in a temperature-controlled (22 ± 2 °C) environment with a 12 h light/dark cycle. Provide the animals with ad libitum access to sterile food and water.
   2. Administer sodium pentobarbital to animals via intraperitoneal injection at a dose of 80 mg/kg for rats or 50 mg/kg for mice to induce anesthesia (see Figure 1).
2. Transcardial perfusion procedure
   1. Fix the anesthetized animals in the supine position on a surgical board.
   2. Cut open the abdominal wall along the midline from the xiphoid process to the pubic symphysis using straight scissors. Cut the lateral thoracic wall bilaterally to fully expose the heart.
   3. Perfuse the animal with ice-cold 0.9% normal saline at a constant flow rate: 100–150 mL over 10–15 min for rats and 20–30 mL over 5–8 min for mice.
3. Brain harvesting and hemisphere separation
   1. Decapitate the animals with large surgical scissors after perfusion completion. Cut the scalp along the midline to expose the skull.
   2. Use a sterile spatula to gently lift the brain from the cranial cavity. Rapidly transfer the whole brain to a sterile Petri dish placed on ice.
   3. Use a sterile, RNase-free razor blade to symmetrically divide the brain into left and right hemispheres along the sagittal suture.
   4. Immediately transfer the left hemisphere to a 15 mL centrifuge tube containing 10 mL of ice-cold 4% paraformaldehyde solution for fixation. Transfer the right hemisphere to a labeled, RNase-free cryopreservation tube, snap-freeze it in liquid nitrogen, and store it at -80 °C until RNA and protein extraction.

2. Brain tissue fixation, sectioning, and long-term storage

1. Fixation and dehydration
   1. Fully immerse the left hemisphere in 4% paraformaldehyde solution and fix the tissue at 4 °C for 24 h. Transfer the brain hemisphere to a 15 mL centrifuge tube containing 30% sucrose solution after completion of fixation, and dehydrate the tissue at 4 °C.
   2. Replace the 30% sucrose solution every 24 h until the brain tissue sinks to the bottom of the tube.
2. Cryosection preparation
   1. Remove the dehydrated brain hemisphere from the sucrose solution and gently blot excess liquid with sterile filter paper.
   2. Mount the brain hemisphere on the cryostat specimen stage with optimal cutting temperature (OCT) embedding medium.
   3. Cut serial coronal brain sections at a thickness of 30 µm using a cryostat. Collect consecutive sections in a 24-well plate containing sterile 0.01 M PBS.
3. Long-term storage of brain sections
   1. Prepare the brain slice cryoprotectant preservation solution in advance: Add 2 g of PVP400, 60 g of sucrose, and 60 mL of ethylene glycol to 100 mL of 0.01 M PBS. Stir the solution at room temperature until all components are fully dissolved and store it at 4 °C until use.

3. RNA extraction with TRIzol

1. Tissue homogenization and phase separation
   1. Homogenize the entorhinal cortical tissue in 1 mL of TRIzol reagent using an ultrafine homogenizer and incubate the homogenate on ice for 15 min. Centrifuge at 8,000 × g for 5 min at 4 °C.
   2. Add 200 µL of chloroform and vortex for 10 s. Centrifuge at 5,600 × g for 15 min at 4 °C. Then transfer the upper aqueous phase to an RNase-free tube. Add 500 µL of isopropanol and incubate at -20 °C overnight to precipitate RNA. Centrifuge again.
2. RNA precipitation, washing, and quality assessment
   1. Discard the supernatant and wash the RNA pellet with 1 mL of 75% ethanol. Centrifuge at 5,600 × g for 5 min at 4 °C. Remove the supernatant, air-dry the pellet for 10 min, and dissolve the RNA in 20 µL of DEPC-treated water for 10 min.
   2. Measure RNA concentration and purity (A₂₆₀/A₂₈₀) using a spectrophotometer. Store the RNA at -80 °C until further analysis.

4. Total protein extraction with Minute kit

1. Tissue lysis and homogenization
   1. Pre-chill the spin column and collection tube on ice. Homogenize the tissue in 100 µL of cell lysis buffer using an ultrafine homogenizer. Rinse the homogenizer with an additional 100 µL of cell lysis buffer and combine the lysates.
2. Protein recovery and quantification
   1. Transfer the homogenate to the spin column and incubate for 2 min at room temperature.
   2. Centrifuge at 11,000–14,300 × g for 2 min.
   3. Place the collection tube on ice immediately after centrifugation and discard the spin column. Determine protein concentration using a BCA assay.

5. Simultaneous RNA and protein extraction from entorhinal cortex (9–10 mg)

1. Pre-experiment preparation and reagent preparation
   1. Use the DNA/RNA/protein kit for all extraction steps. Use only certified RNase-free and DNase-free consumables (see Figure 2).
   2. Prepare the lysis buffer immediately before use: Add protease inhibitor cocktail to the kit-provided lysis buffer at a 1:100 dilution.
2. Tissue lysis and phase separation
   1. Remove the frozen right brain hemisphere from -80 °C storage and place it on ice. Use a sterile RNase-free punch to collect 9–10 mg of tissue from the target brain region.
   2. Homogenize the tissue fully on ice using an electric tissue homogenizer until no visible tissue fragments remain. Incubate the homogenate on ice for 10 min to ensure complete lysis.
   3. Add 200 µL of the kit-provided separation reagent to the homogenate. Vortex the tube vigorously for 30 s and incubate it at room temperature for 3 min. Centrifuge the tube at 8,000 × g for 15 min at 4 °C to achieve complete phase separation.
3. RNA purification and elution
   1. Carefully collect the upper aqueous phase using an RNase-free pipette tip. Transfer the collected aqueous phase to a new 1.5 mL RNase-free tube. Add an equal volume of 100% ethanol to the collected aqueous phase and mix.
   2. Transfer 700 µL of the mixture to the kit-provided RNA purification column. Centrifuge the column at 5,600 × g for 1 min at room temperature.
   3. Add 500 µL of the kit-provided RNA wash buffer 1 to the column. Centrifuge the column at 5,600 × g for 1 min at room temperature.
   4. And add 500 µL of kit-provided RNA wash buffer 2 to the column, centrifuge again. Centrifuge the empty column at 8,000 × g for 2 min at room temperature to completely remove residual ethanol from the column membrane.
   5. Transfer the column to a new RNase-free 1.5 mL microcentrifuge tube. Add 30–50 µL of RNase-free water (preheated to 60 °C) directly to the center of the column membrane. Incubate the column at room temperature for 2 min. Centrifuge the column at 8,000 × g for 1 min at room temperature to elute the RNA.
4. Protein precipitation and dissolution
   1. Add 750 µL of isopropanol to the remaining organic phase and interphase in the original tube after removing the aqueous phase for RNA extraction. Incubate the mixture at -20 °C for 30 min to precipitate proteins.
   2. Centrifuge the tube at 8,000 × g for 10 min at 4 °C. Discard the supernatant and keep the protein pellet intact at the bottom of the tube.
   3. Add 1 mL of 95% ethanol to the protein pellet. Vortex the tube briefly to wash the pellet. Centrifuge the tube at 8,000 × g for 5 min at 4 °C and discard the supernatant. Repeat this wash step two more times.
   4. Air-dry the protein pellet at room temperature for 5–10 min until the pellet becomes translucent and no residual ethanol is visible.
   5. Add 50–100 µL of 5–10% SDS solution to the dried pellet. Vortex the tube vigorously for 1 min and incubate it on ice for 10 min to fully dissolve the protein. Centrifuge the tube at 8,000 × g for 5 min at 4 °C. Collect the supernatant as the final protein extract.

6. Immunohistochemistry (IHC) staining

1. Section pre-treatment and endogenous enzyme inactivation
   1. Remove brain sections from -20 °C cryoprotectant storage. Use a glass needle to transfer the sections to a 24-well plate containing 0.01 M PBS (pH 7.4).
   2. Wash the sections three times with 0.01 M PBS, 5 min per wash, at room temperature on a shaker.
   3. Incubate the sections with 3% hydrogen peroxide peroxidase blocker at room temperature for 10 min to inactivate endogenous peroxidase activity. Repeat the washing steps after blocking.
2. Permeabilization and non-specific blocking
   1. Incubate the sections in 0.5% Triton X-100 for 30 min at room temperature on a shaker for cell membrane permeabilization. Repeat the washing steps.
   2. Incubate the sections in 4% bovine serum albumin for 30 min at room temperature, followed by incubation with a non-specific staining inhibitor for an additional 30 min at room temperature.
3. Primary and secondary antibody incubation
   1. Dilute primary antibodies in 1% BSA solution at the following optimized dilutions: doublecortin (DCX, 1:4000), neuronal nuclear antigen (NeuN, 1:3000), ionized calcium-binding adapter molecule 1 (Iba-1, 1:2000), and glial fibrillary acidic protein (GFAP, 1:2000).
   2. Add 200 µL of diluted primary antibody solution to each well, ensuring full immersion of the sections.
   3. Add 200 µL of biotin-labeled secondary antibody to each well. Incubate the sections at room temperature for 30 min.
   4. Wash the sections three times with 0.01 M PBS, 5 min per wash. Add 300 µL of streptavidin-peroxidase conjugate to each well. Incubate the sections at room temperature for 30 min. Repeat the washing steps after incubation.
4. 3,3′-Diaminobenzidine (DAB) chromogenic development and cover slipping
   1. Add 200 µL of DAB working solution to each well. Monitor the chromogenic reaction under a light microscope.
   2. Mount the sections onto gelatin-coated glass slides using a glass needle. Air-dry the slides completely at room temperature.
   3. Dehydrate the slides through a graded ethanol series (50%, 70%, 90%, 95%, 100%, 100% ethanol, 5 min each), followed by clearance in xylene two times, 5 min each.

7. Immunofluorescence (IF) staining

1. Section pre-treatment and permeabilization
   1. Remove brain sections from -20 °C cryoprotectant storage. Transfer the sections to a 24-well plate containing 0.01 M PBS (pH 7.4).
   2. Wash the sections three times with 0.01 M PBS, 5 min per wash, at room temperature on a shaker.
   3. Incubate the sections in 0.5% Triton X-100 for 30 min at room temperature on a shaker.
   4. Wash the sections three times with 0.01 M PBS, 10 min per wash, after permeabilization.
2. Blocking and primary antibody incubation
   1. Incubate the sections in a blocking solution containing 4% BSA and a non-specific staining inhibitor for 1 h at room temperature in a humidified chamber.
   2. Aspirate the blocking solution. Dilute primary antibodies in 1% BSA solution at optimized concentrations and add 300 µL of the diluted primary antibody solution to each well. Incubate the sections overnight at 4 °C in a humidified, light-proof chamber.
3. Secondary antibody incubation
   1. Rewarm the sections at room temperature for 1 h in the dark the next day. Recover the primary antibody solution and wash the sections three times with 0.01 M PBS, 10 min per wash, on a shaker in the dark.
   2. Dilute fluorophore-conjugated secondary antibodies in 1% BSA solution at the manufacturer's recommended dilution. Add 300 µL of the secondary antibody solution to each well. Incubate the sections at room temperature for 1–2 h in the dark.
   3. Wash the sections five times with 0.01 M PBS, 5 min per wash, in the dark.

8. Quantitative real-time PCR (qPCR) analysis

1. RNA gDNA removal and reverse transcription
   1. Perform reverse transcription to synthesize cDNA using a reverse transcription kit, with 1 µg of total RNA per 20 µL reaction system. Use the manufacturer's recommended thermal cycling program: 25 °C for 5 min, 50 °C for 15 min, 85 °C for 5 min, hold at 4 °C.
   2. Dilute the synthesized cDNA 1:5 with RNase-free water. Store the cDNA at -20 °C until qPCR amplification.
2. qPCR system preparation and amplification
   1. Prepare the qPCR reaction system on ice in a dedicated RNase-free PCR hood. The 20 µL reaction system includes: 10 µL of qPCR Master Mix, 0.4 µL of forward primer (10 µM), 0.4 µL reverse primer (10 µM), 2 µL of diluted cDNA template, 7.2 µL of RNase-free water.
   2. Perform qPCR amplification using the following program: 95 °C for 30 s of initial denaturation; 40 cycles of 95 °C for 10 s and 60 °C for 30 s; followed by dissociation curve analysis from 60 °C to 95 °C, with 0.5 °C increments per 5 s.

9. Western blot

1. Protein quantification and sample preparation
   1. Dilute the protein extracts to equal concentrations using 5–10% SDS solution, based on the BCA assay results.
2. SDS-PAGE electrophoresis and protein transfer
   1. Prepare a 10% SDS-PAGE separating gel and a 5% stacking gel according to standard molecular biology protocols.
   2. Load 20 µg of total protein per well into the gel. For gradient loading experiments, load 15 µg, 30 µg, and 45 µg of protein per well.
   3. Perform electrophoresis at 80 V for 30 min until the protein marker enters the separating gel, then increase the voltage to 120 V for 60–90 min until the bromophenol blue dye reaches the bottom of the gel.
   4. Assemble the transfer sandwich in the following order (from cathode to anode): sponge, filter paper, gel, PVDF membrane, filter paper, and sponge. Ensure that no air bubbles remain between the gel and the membrane.
   5. Perform wet transfer at 300 mA constant current for 90 min in an ice bath.
3. Membrane blocking and antibody incubation
   1. Rinse the PVDF membrane briefly with TBST buffer after transfer completion. Incubate the membrane in 5% BSA for 2 h at room temperature on a shaker to block non-specific binding sites.
   2. Dilute primary antibodies in 5% BSA solution at optimized concentrations. Incubate the membrane with the primary antibody solution overnight at 4 °C on a shaker.
   3. Wash the membrane three times with TBST buffer, 10 min per wash, on a shaker the next day.
   4. Dilute horseradish peroxidase (HRP)-conjugated secondary antibody in 5% non-fat milk solution at the manufacturer's recommended dilution. Incubate the membrane with the secondary antibody solution for 2 h at room temperature on a shaker.
   5. Wash the membrane three times with TBST buffer, 10 min per wash, after secondary antibody incubation. Incubate the membrane with the ECL substrate solution for 1–2 min in the dark.

Results

Assessment of RNA concentration, agarose gel electrophoresis, and qPCR

As shown, total RNA and protein were co-extracted from entorhinal cortex (9–10 mg) punches with the DNA/RNA/protein kit. Parallel samples processed with TRIzol (RNA) or the protein extraction kit served as controls. The protocol yielded 235 ± 12 ng µL⁻¹ RNA, 36 % less than TRIzol (p < 0.05) (Figure 3A). Nevertheless, agarose gels revealed sharp 28S and 18S rRNA bands and 5S signals, indicating intact RNA (Figure 3B). qPCR for CD86, IL-1β, TNF-α, and IL-6 produced single, symmetrical dissociation curves (Figure 3C), lower Ct values were observed in the group (Figure 3D); however, further studies including RNA integrity analyses are required to determine whether these differences reflect improved RNA quality or extraction efficiency . Although RNA yield was lower than that obtained with TRIzol extraction, distinct 28S and 18S rRNA bands and specific qPCR amplification curves indicated that the extracted RNA was of sufficient quality for downstream qPCR analyses.

Evaluation of protein concentration, SDS-PAGE, and Western blot analysis

Consistent with the RNA data, the dual-extraction protocol yielded lower total protein concentrations than the protein kit (p < 0.05). Nevertheless, Western blots revealed markedly stronger immunoreactive bands for every target examined (Figure 4A). Consistent with RNA quantification results, the RNA–protein co-extraction protocol recovers less total protein than the dedicated single-purpose protein kit, which can be observed via CBB whole-protein staining (Figure 4B–C). Housekeeping protein β-Tubulin showed clear and stable bands under both extraction methods, with comparable signal intensity. The low-abundance ULK protein was also successfully detected in co-extracted samples. Notably, the band signals of PSD-95, p-ERK and p-NFκB p65 were markedly stronger in the co-extraction group relative to the single protein extraction kit. Serial protein loading from 15 µg to 45 µg generated dose-dependent band gradients for all detected targets in both groups. Collectively, although the total protein yield of the dual extraction workflow is lower, its protein quality is sufficient for standard Western blot analysis, and overall detection performance is not inferior to conventional single protein extraction kits.

Immunohistochemistry and immunofluorescence

Immunohistochemistry on brain sections stored for 0.5–7 years revealed robust labeling of DCX⁺ newborn neurons, AVP⁺ secretory cells, and MAP2/NeuN⁺ neurons with intact morphology and fibers (Figure 5A). Immunofluorescence of 5–7-year-old sections showed clear GFAP⁺ astrocytes, Iba-1⁺ microglia, and FosB⁺ activated neurons (Figure 5B). Representative sections stored for 0.5–seven years retained detectable immunohistochemical and immunofluorescence signals under the conditions tested. Thus, the preservation solution maintains tissue integrity and antigenicity, enabling reliable long-term storage.

Figure 1: Hemisphere separation integrated workflow for brain morphological and molecular detection. This schematic illustrates the full experimental pipeline, including rodent transcardial saline perfusion, midline bisection of the isolated whole brain, fixation and cryosection preparation of the left hemisphere for IHC/IF staining, and cryopreservation of the contralateral hemisphere for subsequent simultaneous RNA-protein co-extraction, qPCR, and Western blot analysis. All mouse illustrations were originally generated by Jimeng AI. Please click here to view a larger version of this figure.

Figure 2: Workflow for simultaneous RNA and protein co-extraction from frozen rodent brain tissue. The diagram displays sequential steps covering target tissue punching from frozen hemispheres, tissue lysis and three-phase separation, RNA purification and elution for qPCR, as well as protein precipitation, washing and dissolution to prepare samples for Western blot detection. Please click here to view a larger version of this figure.

Figure 3: Evaluation of RNA yield, RNA integrity, and qPCR compatibility using microscale rat entorhinal cortex samples. (A) Quantified RNA concentrations obtained via co-extraction kit versus TRIzol method (mean ± SEM). (B) Agarose gel electrophoresis showing intact 28S and 18S rRNA bands from both extraction groups. (C) Single melting curves confirm specific amplification of target inflammatory genes and GAPDH as the reference gene. (D) Ct values of detected inflammatory genes under identical RNA input across two extraction methods. *p < 0.05, **p < 0.01 vs TRIzol group. Please click here to view a larger version of this figure.

Figure 4: Western blot assessment of proteins recovered by the RNA/protein co-extraction protocol. (A) Gradient protein loading (15/30/45 µg) showing dose-dependent band signals of synaptic and signaling proteins. (B) ULK1 large molecular weight protein detection with CBB total protein staining as loading reference. (C) Equal-loading Western blot comparison of target proteins between two extraction workflows. Please click here to view a larger version of this figure.

Figure 5: Immunohistochemical and immunofluorescent staining of free-floating brain sections preserved for 0.5–7 years. (A) Brightfield immunohistochemical staining micrographs captured at 200× and 400× magnification. DCX and AVP staining was performed on mouse tissue samples, whereas MAP2 and NeuN staining was performed on rat tissue samples. (B) Representative immunofluorescence images obtained from rat tissue samples. GFAP labels astrocytes, IBA-1 labels microglia, and FOSB indicates neuronal activity. All immunofluorescence staining experiments shown in this panel were conducted using rat samples to evaluate neuronal marker expression and cellular localization, micrographs at 200× magnification.  Please click here to view a larger version of this figure.

Discussion

In murine research, morphological analyses and nucleic acid/protein assays often require separate animal samples because of their distinct preparation procedures. This increases animal use and introduces inter-animal biological variation, which may reduce comparability between histological and molecular datasets. To address these limitations, we developed a workflow that enables histological and molecular data to be obtained from the same animal.

The central design of this workflow is the hemispheric allocation of brain tissue after transcardial perfusion with 0.9% saline^5,6. One hemisphere is fixed in 4% paraformaldehyde for immunohistochemistry and immunofluorescence, whereas the contralateral hemisphere is stored at −80 °C for subsequent RNA–protein co-extraction, qPCR, and Western blotting. However, the selected readouts may not be fully comparable between the two cerebral hemispheres. Structural and functional lateralization has been documented in rodent brains, and such hemispheric differences may introduce variability when analyses are performed on opposite sides^7,8. Therefore, researchers should consistently select tissue from the same cerebral hemisphere in accordance with their research aims.

A prominent advantage of this method is the long-term preservation of fixed, floating brain sections. Following dehydration with 30% sucrose, 30 µm coronal sections are stored in a sucrose-ethylene glycol-PVP preservation solution. Previous studies have shown that sucrose–ethylene glycol–PVP cryoprotectant solutions help preserve tissue morphology and antigenicity during long-term storage^9,10. However, few studies have evaluated storage durations extending over several years. Our laboratory data extend these findings: representative sections stored for up to seven years retained detectable immunohistochemical and immunofluorescence signals under the tested conditions. These observations support the feasibility of storing floating brain sections for several years for retrospective analyses and follow-up experiments.

Notably, this workflow offers another key advantage through dual RNA–protein co-extraction from a single frozen brain hemisphere. Although this co-extraction protocol yields slightly lower RNA and protein quantities than conventional single-target extraction methods, the obtained biomaterials are still sufficient for robust qPCR and Western blot detection. Critically, co-extraction allows transcript and protein abundance to be quantified from identical tissue samples, eliminating biological variation arising from separate specimen processing and enabling highly consistent, integrated molecular profiling^11,12. Furthermore, this approach requires only a small tissue mass of approximately 9–10 mg, making it well-suited for analyses of discrete, region-specific brain subpopulations with limited sample availability.

Despite these significant advantages, this method also has certain limitations that warrant acknowledgment. First, the relatively low RNA and protein yields may restrict its applicability for experiments requiring large quantities of nucleic acids or proteins, such as large-scale protein purification or high-depth RNA sequencing. Second, while long-term section storage is effective for up to seven years, gradual degradation of certain labile antigens or nucleic acids may still occur over extended periods, which could compromise staining intensity or molecular detection sensitivity. Third, the method is currently optimized for murine brain tissue and may require modification for application to other tissues or alternative animal models, as tissue composition and structural characteristics vary across species and organs. Finally, 5–10% SDS may interfere with mass spectrometry, enzyme activity assays, protein–protein interaction studies^13,14, and other SDS-sensitive applications. Additional detergent removal or buffer exchange procedures may therefore be required.

In conclusion, this hemispheric allocation workflow enables paired morphological and molecular analyses from the same animal while reducing animal use and inter-animal variation. This workflow would be useful in studies requiring paired histological and molecular analyses, particularly when tissue availability is limited. Its long-term section storage capability may also facilitate retrospective analyses and standardized sample archiving, while the use of small brain-region samples supports region-specific investigations. Its main limitations are lower RNA and protein yields, potential hemispheric asymmetry, restriction to murine brain tissue, and limited compatibility with SDS-sensitive applications. Future studies should optimize biomolecule recovery, validate the workflow in other tissues and species, and assess the long-term stability of additional molecular targets.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL RNase-free microcentrifuge tubes	Axygen	MCT-150-C-S	Collection of tissue punches and RNA/protein extraction fractions.
15 mL conical centrifuge tubes	Biofil	CFT010150	Fixation, sucrose dehydration, and solution storage.
24-well cell culture plates	NEST	702001	Collection, staining, and long-term storage of free-floating brain sections.
30% sucrose solution	Prepared in laboratory	N/A	Cryoprotection of fixed brain hemispheres before cryosectioning.
4% paraformaldehyde solution	Biosharp	BL539A	Immersion fixation of the morphology-designated brain hemisphere.
5× protein loading buffer	Beyotime Biotechnology	DL101	Preparation of protein samples for SDS-PAGE.
Agarose	Solarbio	Cat: A8201	RNA integrity assessment by agarose gel electrophoresis.
Anti-doublecortin antibody	Abcam	ab18723	Primary antibody for doublecortin immunohistochemistry.
Anti-ERK antibody	Beyotime	AB2014	Primary antibody for total ERK Western blotting.
Anti-GFAP antibody	Abcam	ab10062	Primary antibody for glial fibrillary acidic protein immunofluorescence.
Anti-Iba1 antibody	Wako	NCNP24	Primary antibody for ionized calcium-binding adaptor molecule 1 immunofluorescence.
Anti-NeuN antibody	Millipore	A60	Primary antibody for neuronal nuclear antigen staining.
Anti-NF-κB p65 antibody	HUABIO	ET1603-12	Primary antibody for NF-κB p65 Western blotting.
Anti-phospho-ERK antibody	Beyotime	ET1610-13	Primary antibody for phosphorylated ERK Western blotting.
Anti-PSD95 antibody	HUABIO	ET1602-20	Primary antibody for PSD95 Western blotting.
Anti-ULK1 antibody	HUABIO	ET1704-63	Primary antibody for ULK1 Western blotting.
Anti-β-tubulin antibody	ABclonal	AC021	Loading-control primary antibody for Western blotting.
Antifade fluorescence mounting medium	Beyotime	P0126-5	Mounting medium for immunofluorescence-stained sections.
Biotin-streptavidin peroxidase detection kit	Fuzhou Maixin Biotech Co., Ltd.	KIT-9720	Secondary detection system for DAB immunohistochemistry.
Bovine serum albumin	Qiansheng Biotech Co., Ltd.	QO3000	Blocking buffer for immunostaining and Western blotting.
C57BL/6 mice	Liaoning Changsheng Biotechology Co.,LTD	4-week-old C57BL/6 mice	Experimental animals for mouse brain tissue collection, if included.
Chemiluminescence imaging system	Clinx Science Instruments Co., Ltd.	ChemiScope 6200 Touch	Detection of Western blot chemiluminescent signals.
Confocal laser scanning microscope	Leica Microsystems CMS GmbH	SN: 591974	Image acquisition for immunofluorescence sections.
Cryoprotectant preservation solution	Prepared in laboratory	N/A	Long-term storage of free-floating brain sections at −20 °C.
Cryostat	Leica	CM1950	Preparation of 30 μm coronal brain sections.
DAB chromogenic substrate kit	Sigma	SLBP7387V	Chromogenic development for immunohistochemistry.
DAPI nuclear staining solution	Coolaber	SL7100	Nuclear counterstain for immunofluorescence.
Digital light microscope	Ningbo Yongxin Optics Co., Ltd.	Model NE910+FL-900	Monitoring DAB development and imaging bright-field-stained sections.
DNase I	Vazyme	R223-01	Removal of residual genomic DNA before reverse transcription.
Donkey anti-rabbit IgG, 488-conjugated	Thermo Fisher Scientific	A21206	Fluorescent secondary antibody for immunofluorescence.
Donkey anti-rabbit IgG, 594-conjugated	Abcam	ab150076	Fluorescent secondary antibody for immunofluorescence.
E.Z.N.A. DNA/RNA/Protein Kit	Omega Bio-Tek	D3892-01
Electrophoresis power supply	Bio-Rad	1645050	Power supply for agarose gel electrophoresis and SDS-PAGE.
Ethylene glycol	Sinopharm Chemical Reagent Co.Ltd.	10009828	Component of cryoprotectant preservation solution.
Gel imaging system	Clinx Science Instruments Co., Ltd.	ChemiScope 6200 Touch	Imaging of agarose gels for RNA quality evaluation.
Gelatin-coated glass slides	CITOTEST LABWARE MANUFACTURING CO.,LTD	REF: 10127105P P/N: 80312-2101	Mounting DAB-stained free-floating brain sections.
Image analysis software	National Institutes of Health	ImageJ/Fiji	Densitometric analysis of Western blot bands and image processing.
Isopropanol	Sinopharm Chemical Reagent Co.Ltd.	80109218	Protein precipitation from the organic/interphase fraction.
Microcentrifuge, refrigerated	Yancheng Kaite Laboratory Instrument Co., Ltd.	TGL16M	Centrifugation during RNA/protein extraction and washing steps.
Minute Protein Extraction Kit	Invent	MS-026
Neutral balsam mounting medium	Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China	Catalog No. S30509, CAS 96949-21-2	Coverslipping of dehydrated DAB-stained sections.
OCT embedding medium	Sakura	4583	Embedding medium for cryosectioning fixed brain hemispheres.
Optical qPCR plates or tubes	Axygen	PCR-0208-C	Reaction vessels for qPCR amplification.
Parafilm	amcor	PM996	Sealing multiwell plates for long-term section storage.
Pentobarbital sodium	Hualan Bio	57-33-0	Anesthetic agent before transcardial perfusion.
Petri dishes	Biosharp	BS-90-D	On-ice brain handling and hemisphere separation.
Phenol/guanidinium RNA extraction reagent	Sparklade	AC0101-B	Comparator reagent for RNA extraction control experiments.
Phosphatase inhibitor cocktail	APExBIO	K1015-100	Preservation of phosphorylation during protein extraction.
Phosphate-buffered saline	Biosharp	BL601A	Washing buffer, sucrose solution solvent, and cryoprotectant solvent.
Polyvinylidene fluoride membrane	Millipore	IPVH08100	Membrane for Western blot transfer.
Polyvinylpyrrolidone K40	Sigma	9003-39-8	Component of cryoprotectant preservation solution.
Protease inhibitor cocktail	Beyotime	P1045	Prevention of protein degradation during tissue lysis.
Protein extraction kit	Invent	MS-026	Comparator kit for total protein extraction control experiments.
Real-time PCR system	Amolarray	MA6000	Quantitative PCR amplification and melt-curve analysis.
Sodium dodecyl sulfate	BioFroxx	3250GR500	Protein pellet dissolution and SDS-PAGE sample preparation.
Sterile razor blades	Biosharp	BS-RB-11	Hemisphere separation and tissue trimming on ice.
Streptavidin-peroxidase conjugate	Fuzhou Maixin Biotech Co., Ltd.	KIT-9720	Signal amplification/detection reagent for DAB immunohistochemistry.
Sucrose	Hushi	57-50-1	Cryoprotection solution and cryoprotectant preservation solution.
SYBR qPCR master mix	Vazyme	Q711-02	Quantitative PCR amplification.
Transfer apparatus	Bio-Rad	1703930	Wet transfer of proteins from SDS-PAGE gel to membrane.
Tris-buffered saline with Tween 20	Prepared in laboratory	N/A	Western blot membrane washing buffer.
Triton X-100	Sinopharm	9002-93-1	Permeabilization of brain sections for immunostaining.
Trizol	Sparklade	AC0101-B
UltraSensitive SP Immunohistochemistry Detection Kit (for mouse/rabbit primary antibody)	Fuzhou Maixin Biotech Co., Ltd.	KIT-9720	Secondary antibody/detection reagent for DAB immunohistochemistry.
Wistar rat	Liaoning Changsheng Biotechology Co.,LTD	280-320 g, male	Experimental animals for rat brain tissue collection, if included.
Xylene	Sinopharm Chemical Reagent Co.Ltd.	10023418	Clearing of DAB-stained slides before coverslipping.
Neutral balsam mounting medium	Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China	Catalog No. S30509, CAS 96949-21-2	Coverslipping of dehydrated DAB-stained sections.
Streptavidin-peroxidase conjugate	Fuzhou Maixin Biotech Co., Ltd.	KIT-9720	Signal amplification/detection reagent for DAB immunohistochemistry.
Triton X-100	Sinopharm	9002-93-1	Permeabilization of brain sections for immunostaining.
UltraSensitive SP Immunohistochemistry Detection Kit (for mouse/rabbit primary antibody)	Fuzhou Maixin Biotech Co., Ltd.	KIT-9720	Secondary antibody/detection reagent for DAB immunohistochemistry.
Xylene	Sinopharm Chemical Reagent Co.Ltd.	10023418	Clearing of DAB-stained slides before coverslipping.