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 principle....

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. Meanw....

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 18.......

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 hemi.......

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.