The authors would like to thank Ikiel Ptito for his continued technical support.

Part 1: Pre-processing of tissue
<ol>
<li>Tissue should be well perfused with paraformaldehyde, glutaraldehyde, or formalin. This can be achieved through standard transcardial perfusion typically used to harvest other organs. In the present study the subject was deeply sedated with ketamine hydrochloride (10 mg/kg, i.m.), euthanized with an overdose of sodium pentobarbital (25 mg/kg, i.v.) and perfused transcardially with 0.1 M PBS until completely exsanguinated. This is followed by a 4% paraformaldehyde solution in PBS for 5 min (~1 liter).</li>
<li>The brain should be stereotaxically blocked, removed from the skull, weighed, and volume determined2. The tissue should then be cryoprotected in graded sucrose solutions with a final concentration of 30% sucrose in phosphate buffer. Blocks of tissue should then be frozen in isopentane at -65&deg;C and stored at -80&deg;C until you are ready to slice. </li>
</ol>
Part 2: Systematic Sampling
<ol>
<li>Systematic sampling relies on a proper plan prior to sectioning. The entire region of interest in both the rostral-caudal and dorsal-ventral extents should be well defined. A stereotaxic atlas of the species in use is helpful to define the region of interest and to determine the length of the region of interest. Once the length of the region of interest is determined it is necessary to establish the sampling interval. For example, from the stereotaxic atlas you determine that the entire rostro-caudal extent of the region of interest is approximately 3mm in length when sectioning in the coronal plane. You then decide to set the cryostat to section at 50&mu;m, at this rate you will have 60 sections covering the rostral-caudal extent of the region of interest. For a typical unbiased stereological study 6-10 systematically sampled sections are required to obtain reliable results. In this example 10 sections equally spaced throughout the rostro-caudal extent result in a sampling interval of 1/6 sections. </li>
</ol>
Part 3: Creation of the brain bank
<ol>
<li>Once the section sampling frequency has been determined, the next step is to determine which stains will be performed immediately. For example, if you are staining with cresyl violet you will capture 1/6 sections on a slide. This will be series 1. If you know there maybe several potential antibodies that you would like run, but either want to see the data that your primary stain provides or simply do not have the time to perform the immunohistochemistry, place the remaining 5 series of sections in the order that they were sliced into wells containing antigen preserve (1% polyvinyl pyrrolidone, 50% ethylene glycol in o.1M PBS, Ph 7.4). The table below provides a sampling scheme consisting of 1 slide and 1 plate (standard 48-well plate) of sections as part of a brain bank (Table 1). <img src="/files/ftp_upload/1260/table1.jpg" alt="Table 1" /> Table 1. The section sampling frequency here is 1/6. The first section is placed on a slide for cresyl violet staining and the next 5 sections are placed into antigen preserve. Section number 7 is then captured on the slide and sections 9-12 are placed in antigen preserve. This cycle is repeated until the region of interest or block of tissue is exhaustively sectioned.</li>
<li>The next step is to exhaustively section the block of tissue. Place a small amount of embedding medium on the chuck and let it freeze. Place the chuck into the microtome head and shave off enough of the frozen medium to have a flat surface. Remove the chuck from the microtome head and place flat within the cryostat. Pour embedding medium on the chuck and firmly place the brain block with the stereotaxically cut side positioned flatly on the chuck. Slowly and completely embed the brain in mounting medium. Place the chuck with the brain into the microtome head and section using the pre-determined parameters for the section sampling frequency.</li>
<li>Once the block of tissue has been exhaustively sectioned and sections are placed into wells, cover the plate with the lid, wrap the lid with parafilm, and place in a -20&deg;C freezer. Log the total number of sections taken for each series, the section-sampling interval, and the number of series for each animal. This log will be vital to keep track of subsequent removal of sections from the brain bank for future immunohistochemistry.</li>
</ol>
Part 4: Representative Results:
Systematical sampling in this manner has been a standard practice in our laboratory for the past 3 years. We have had a great deal of success performing immunohistochemistry on material that has been stored in antigen preserve three years after it was sliced without deterioration of the signal (Figure 1). Furthermore, as part of our brain bank we have logged close to 20,000 systematic sections of the non-human primate brain (Figure 2) as part of our long-term research plans. <img src="/files/ftp_upload/1260/1260figure1.jpg" alt="" /> Figure 1. This is a section from the occipital pole of the non-human primate immunostained for NeuN showing both layer VI and interstitial white matter neurons. This particular section was stored in antigen preserve at -20&deg;C for a period of 2 years. Scale bar = 100&micro;m. <img src="/files/ftp_upload/1260/1260fig2.jpg" alt="figure2" /> Figure 2. Our vervet brain bank now consists of close to 20,000 systematic sections from over 30 monkeys.

<ol>
	<li>West, M., Slomianka, L., Gundersen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unbiased+stereological+estimation+of+the+total+number+of+neurons+in+the+subdivisions+of+the+rat+hippocampus+using+the+optical+fractionator.+Aat+Rec.">Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator. Aat Rec.</a> 231, 482-497 (1991).</li><li>Burke, M. W., Zangenehpour, S., Boire, D., Ptito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+Non-human+Primate+Brain+in+Stereotaxic+Space.">Dissecting the Non-human Primate Brain in Stereotaxic Space.</a> J Vis Exp. , (2009).</li></ol>

The authors have nothing to disclose.

Systematic sampling is an inexpensive method intended to maximize research material. This sampling strategy is designed to comply with the rules of unbiased stereology that requires systematic sampling throughout the region. It is critical that the order of sections for each series is maintained. In our laboratory we have successfully used this method for banking both hamster and non-human primate brain sections. So far, we have collected close to 20,000 vervet monkey (Chlorocebus aethiops sabeus) brain sections...

<table><tbody><tr><td>Ethylene glycol</td><td>Fisher Scientific</td><td>E178-4</td><td></td></tr><tr><td>Polyvinyl pyrrolidone</td><td>Fisher Scientific</td><td>BP431-500</td><td></td></tr><tr><td>Thermal Scientific Embedding Medium</td><td>Fisher Scientific</td><td></td><td></td></tr></tbody></table>

brain banking: making the most of your research specimens

Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given area of interest1. To achieve this goal 6-10 systematic sections should be probed covering the entire structure. Typically this involves processing 1/5 sections which leaves a significant amount of material unprocessed. In order to maximize the material, we propose an inexpensive method for preserving fixed tissue as part of a long-term storage research plan. As tissue is sliced and processed for the desired stain or antibody, alternate sections should be systematically placed in antigen preserve at -20&deg;C for future processing. Using 24-well plates, sections can be placed in order for future retrieval. Using this method, tissue can be stored and processed for immunohistochemistry over the course of years.

Watch this Scientific Journal Video about Brain Banking: Making the Most of your Research Specimens at JoVE.com

Brain Banking: Making the Most of your Research Specimens

Brain banking and systematic sampling of biological material provides the basis for unbiased stereology and maximizes the potential data obtained from each specimen.

Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given area of interest1.  To ...

brain-banking-making-the-most-of-your-research-specimens

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.9K Views.  University of Montreal. Brain banking and systematic sampling of biological material provides the basis for unbiased stereology and maximizes the potential data obtained from each specimen.

Video: Brain Banking: Making the Most of your Research Specimens

Creation and Maintenance of a Living Biobank - How We Do It

In light of the growing knowledge about the inter-individual properties and heterogeneity of cancers, the emerging field of personalized medicine requires a platform for preclinical research. Over recent years, we have established a biobank of colorectal and pancreatic cancers comprising of primary tumor tissue, normal tissue, sera, isolated peripheral blood lymphocytes (PBL), patient-derived xenografts (PDX), as well as primary and secondary cancer cell lines. Since original tumor tissue is limited and the establishment rate of primary cancer cell lines is still relatively low, PDX allow not only the preservation and extension of the biobank but also the generation of secondary cancer cell lines. Moreover, PDX-models have been proven to be the ideal in vivo model for preclinical drug testing. However, biobanking requires careful preparation, strict guidelines and a well attuned infrastructure. Colectomy, duodenopancreatectomy or resected metastases specimens are collected immediately after resection and transferred to the pathology department. Respecting priority of an unbiased histopathological report, at the discretion of the attending pathologist who carries out the dissections, small tumor pieces and non-tumor tissue are harvested. 
Necrotic parts are discarded and the remaining tumor tissue is cut into small, identical cubes and cryopreserved for later use. Additionally, a small portion of the tumor is minced and strained for primary cancer cell culture. Additionally, blood samples drawn from the patient pre- and postoperatively, are processed to obtain serum and PBLs. For PDX engraftment, the cryopreserved specimens are defrosted and implanted subcutaneously into the flanks of immunodeficient mice. The resulting PDX closely recapitulate the histology of the "donor" tumors and can be either used for subsequent xenografting or cryopreserved for later use. In the following work, we describe the individual steps of creation, maintenance and administration of a large biobank of colorectal and pancreatic cancer. Moreover, we highlight the crucial details and caveats associated with biobanking.

In the following work, we describe the consecutive steps necessary for the establishment of a large biobank of colorectal and pancreatic cancer.

In light of the growing knowledge about the inter-individual properties and heterogeneity of cancers, the emerging field of personalized medicine requires ...

Cancer Research

Biobank for Translational Medicine: Standard Operating Procedures for Optimal Sample Management

Biobanks are key research infrastructures aimed at the collection, storage, processing, and sharing of high-quality human biological samples and associated data for research, diagnosis, and personalized medicine. The Biobank for Translational and Digital Medicine Unit at the European Institute of Oncology (IEO) is a landmark in this field. Biobanks collaborate with clinical divisions, internal and external research groups, and industry, supporting patients' treatment and scientific progress, including innovative diagnostics, biomarker discovery, and clinical trial design. Given the central role of biobanks in modern research, biobanking standard operating procedures (SOPs) should be extremely precise. SOPs and controls by certified specialists ensure the highest quality of samples for the implementation of science-based, diagnostic, prognostic, and therapeutic personalized strategies. However, despite numerous efforts to standardize and harmonize biobanks, these protocols, which follow a strict set of rules, quality controls, and guidelines based on ethical and legal principles, are not easily accessible. This paper presents the biobank standard operating procedures of a large cancer center.

Biobanks are crucial resources for biomedical research and the Biobank for Translational and Digital Medicine Unit at the European Institute of Oncology is a model in this field. Here, we provide a detailed description of biobanks' standard operating procedures for the management of different types of human biological samples.

Biobanks are key research infrastructures aimed at the collection, storage, processing, and sharing of high-quality human biological samples and ...

Protocol for Prostatectomy Specimen Mapping and Dissection

Enhancing Prostate Tumor Biobanking Reliability with Improved Sampling Technique and Histological Characterization

Acquiring fresh and well-characterized tumor tissue samples is critical for conducting high-quality &#34;omics&#34; studies. However, it can be particularly challenging in the context of prostate cancer (PC) due to the unique nature of this organ and the high heterogeneity associated with this tumor. On the other hand, histopathologically characterizing samples before their storage without causing significant tissue alterations is also an intriguing challenge. In this context, we present a new method for acquiring, mapping, characterizing, and micro-dissecting resected prostate tissue based on anatomopathological criteria.
Unlike previously published protocols, this method reduces the time required for histopathological analysis of the prostate specimen without compromising its structure, which is crucial for assessing surgical margins. Furthermore, it enables the delineation and micro-macro dissection of fresh prostate tissue samples, with a focus on histological tumor areas defined by pathological criteria such as Gleason score, precursor lesions (high-grade prostatic intraepithelial neoplasia - PIN), and inflammatory lesions (prostatitis). These samples are then stored in a Biobank for subsequent research analyses.

Author Spotlight: Advancing Prostate Cancer Research Through Improved Tissue Sampling and Biobanking

This protocol describes a method for facilitating the collection of samples from radical prostatectomy specimens. The goal is to map, characterize, and micro-macro dissect tissue samples from the specimens based on anatomopathological criteria before storing them in a Biobank.

Acquiring fresh and well-characterized tumor tissue samples is critical for conducting high-quality &#34;omics&#34; studies. However, it can be ...

Symmetric Bihemispheric Postmortem Brain Cutting to Study Healthy and Pathological Brain Conditions in Humans

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena described in those patients for whom a brain autopsy has been requested. Although the advancements of biomedical sciences and neuroimaging have revolutionized the neuropsychiatric field, they have also generated the misleading idea that brain autopsies have only a confirmatory value. This false idea created a drastic reduction of autopsy rates and, consequently, a reduced possibility to perform more detailed and extensive neuropathological investigations, which are necessary to comprehend numerous normal and pathological aspects yet unknown of the human brain. The traditional inferential method of correlation between observed neuropsychiatric phenomena and corresponding localization/characterization of their possible neurohistological correlates continues to have an undeniable value. In the context of neuropsychiatric diseases, the traditional clinicopathological method is still the best possible methodology (and often the only available) to link unique neuropsychiatric features to their corresponding neuropathological substrates, since it relies specifically upon the direct physical assessment of brain tissues. The assessment of postmortem brains is based on brain cutting procedures that vary across different neuropathology centers. Brain cuttings are performed in a relatively extensive and systematic way based on the various clinical and academic contingencies present in each institution. A more anatomically inclusive and symmetric bi-hemispheric brain cutting methodology should at least be used for research purposes in human neuropathology to coherently investigate, in depth, normal and pathological conditions with the peculiarities of the human brain (i.e., hemispheric specialization and lateralization for specific functions). Such a method would provide a more comprehensive collection of neuropathologically well-characterized brains available for current and future biotechnological and neuroimaging techniques. We describe a symmetric bi-hemispheric brain cutting procedure for the investigation of hemispheric differences in human brain pathologies and for use with current as well as future biomolecular/neuroimaging techniques.

Organized brain cutting procedures are necessary to correlate specific neuropsychiatric phenomena with definitive neuropathologic diagnoses. Brain cuttings are performed differently based on various clinico-academic contingencies. This protocol describes a symmetric bihemispheric brain cutting procedure to investigate hemispheric differences in human brain pathologies and to maximize current and future biomolecular/neuroimaging techniques.

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena ...

Medicine

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into extracellular plaques of the brain. The A&#946; peptides, composed of 40 amino acids, are generated from amyloid precursor proteins (APP) by &#946;- and &#947;-secretases. A&#946; is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of A&#946; peptides were identified, the detailed production and distribution of individual A&#946; peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 &#181;m. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various A&#946; species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 &#181;m) imaging of an AD brain and severe CAA sample clearly shows that A&#946;1-36 to A&#946;1-41 were deposited into leptomeningeal vessels, while A&#946;1-42 and A&#946;1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid &#946; protein on brain tissues of Alzheimer&#39;s disease and cerebral amyloid angiopathy samples using MALDI-IMS.

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into ...

Neuroscience

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...

Lipidomics and Transcriptomics in Neurological Diseases

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with various signaling or ligand function that can underscore the onset and progression of diseases. Often changing at the presymptomatic level, lipids are an emerging source of drug targets and biomarkers. Many neurological diseases exhibit neuroinflammation, neurodegeneration, and neuronal excitability as common hallmarks, partly modulated by specific lipid signaling systems. The interdependence and interrelation of synthesis of various lipids prompts a multilipid, multienzyme, and multireceptor analysis in order to derive the commonalities and specificities of neurological contexts and to expedite the unravelling of mechanistic aspects of disease onset and progression. Ascribing lipid roles to distinct brain regions advances the determination of lipid molecular phenotype and morphology associated with a neurological disease.
Presented here is a modular protocol suitable for the analysis of membrane lipids and downstream lipid signals along with mRNA of enzymes and mediators underlying their functionality, extracted from discrete brain regions that are relevant for a particular neurological disease and/or condition. To ensure accurate comparative lipidomic profiling, the workflows and operating criteria were optimized and standardized for: i) brain sampling and dissection of regions of interest, ii) co-extraction of multiple lipid signals and membrane lipids, iii) dual lipid/mRNA extraction, iv) quantification by liquid chromatography multiple reaction monitoring (LC/MRM), and v) standard mRNA profiling. This workflow is amenable for the low tissue amounts obtained by sampling of the functionally discrete brain subregions (i.e. by brain punching), thus preventing bias in multimolecular analysis due to tissue heterogeneity and/or animal variability. To reveal peripheral consequences of neurological diseases and establish translational molecular readouts of neurological disease states, peripheral organ sampling, processing, and their subsequent lipidomic analysis, as well as plasma lipidomics, are also pursued and described. The protocol is demonstrated on an acute epilepsy mouse model.

This article presents a modular protocol for tissue lipidomics and transcriptomics, and plasma lipidomics in neurological disease mouse models targeting lipids underlying inflammation and neuronal activity, membrane lipids, downstream messengers, and mRNA-encoding enzymes/receptors underlying lipid function. Sampling, sample processing, extraction, and quantification procedures are outlined.

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with ...

Comprehensive Autopsy Program for Individuals with Multiple Sclerosis

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 days a year. Participants consent to donate their brain and spinal cord. Most patients were followed by neurologists at the Cleveland Clinic Mellen Center for MS Treatment and Research. Their clinical courses and neurological disabilities are well-characterized. Soon after death, the body is transported to the MS Imaging Center, where the brain is scanned in situ by 3 T magnetic resonance imaging (MRI). The body is then transferred to the autopsy room, where the brain and spinal cord are removed. The brain is divided into two hemispheres. One hemisphere is immediately placed in a slicing box and alternate 1 cm-thick slices are either fixed in 4% paraformaldehyde for two days or rapidly frozen in dry ice and 2-methylbutane. The short-fixed brain slices are stored in a cryopreservation solution and used for histological analyses and immunocytochemical detection of sensitive antigens. Frozen slices are stored at -80 &#176;C and used for molecular, immunocytochemical, and in situ hybridization/RNA scope studies. The other hemisphere is placed in 4% paraformaldehyde for several months, placed in the slicing box, re-scanned in the 3 T magnetic resonance (MR) scanner and sliced into centimeter-thick slices. Postmortem in situ MR images (MRIs) are co-registered with 1 cm-thick brain slices to facilitate MRI-pathology correlations. All brain slices are photographed and brain white-matter lesions are identified. The spinal cord is cut into 2 cm segments. Alternate segments are fixed in 4% paraformaldehyde or rapidly frozen. The rapid procurement of postmortem MS tissues allows pathological and molecular analyses of MS brains and spinal cords and pathological correlations of brain MRI abnormalities. The quality of these rapidly-processed postmortem tissues (usually within 6 h of death) is of great value to MS research and has resulted in many high-impact discoveries.

Multiple sclerosis is an inflammatory demyelinating disease with no cure. Analysis of brain tissue provides important clues to understanding the pathogenesis of the disease. Here we discuss the methodology and downstream analysis of MS brain tissue collected through a unique rapid autopsy program in operation at the Cleveland Clinic.

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 ...

Abbiategrasso Brain Bank Protocol for Collecting, Processing and Characterizing Aging Brains

In a constantly aging population, the prevalence of neurodegenerative disorders is expected to rise. Understanding disease mechanisms is the key to find preventive and curative measures. The most effective way to achieve this is through direct examination of diseased and healthy brain tissue. The authors present a protocol to obtain, process, characterize and store good quality brain tissue donated by individuals registered in an antemortem brain donation program. The donation program includes a face-to-face empathic approach to people, a collection of complementary clinical, biological, social and lifestyle information and serial multi-dimensional assessments over time to track individual trajectories of normal aging and cognitive decline. Since many neurological diseases are asymmetrical, our brain bank offers a unique protocol for slicing fresh specimens. Brain sections of both hemispheres are alternately frozen (at -80 &#176;C) or fixed in formalin; a fixed slice on one hemisphere corresponds to a frozen one on the other hemisphere. With this approach, a complete histological characterization of all frozen material can be obtained, and omics studies can be performed on histologically well-defined tissues from both hemispheres thus offering a more complete assessment of neurodegenerative disease mechanisms. Correct and definite diagnosis of these diseases can only be achieved by combining the clinical syndrome with the neuropathological evaluation, which often adds important etiological clues necessary to interpret the pathogenesis. This method can be time consuming, expensive and limited as it only covers a limited geographical area. Regardless of its limitations, the high degree of characterization it provides can be rewarding. Our ultimate goal is to establish the first Italian Brain Bank, all the while emphasizing the importance of neuropathologically verified epidemiological studies.

This protocol describes a method to track individual brain-aging trajectories through a brain donation program and proper characterization of brains. Brain donors are involved in a long-term longitudinal study including serial multi-dimensional assessments. The protocol contains a detailed description of brain processing and an accurate diagnostic methodology.

In a constantly aging population, the prevalence of neurodegenerative disorders is expected to rise. Understanding disease mechanisms is the key to find ...

Collection of Frozen Rodent Brain Regions for Downstream Analyses

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5&#8211;1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 &#176;C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...