Rodent Brain Tissue Research Articles

Evaluating blood-brain barrier disruption and infarction volume concurrently in rats subjected to ischemic stroke using an optical imaging system

BackgroundBlood-brain barrier (BBB) disruption is pivotal in the pathophysiological process of ischemic stroke and is often measured in rodent stroke studies. Traditionally, rodent BBB permeability increase is determined by measuring cerebral leakage of certain dyes such as Evans Blue or sodium fluorescein (NaFL). However, due to the special processing of samples for BBB permeability measurement, they cannot be used afterward for determining other essential parameters such as cerebral infarction volume. Therefore, using different batches of animals for assessing BBB permeability and infarction volume is typical. However, this would limit the stroke study’s statistical power and scientific value while hindering the implementation of procedures for high standard animal welfare. New methodThe rats subjected to middle cerebral artery occlusion (MCAO) were intraperitoneally injected with NaFL during the reperfusion phase. The brains were sliced and measured for BBB permeability using the small animal optical imaging system (IVIS® Lumia series III). Afterward, the same brain samples were either sliced or homogenized for tests that assessed infarction volume or other molecular changes. ResultsThe sum fluorescence intensity of the ischemic brain slices under the IVIS® Lumia series Ⅲ showed a strong correlation with the infarction volume determined by 2,3,5-triphenyl tetrazolium chloride (TTC) staining (r = 0.7440, P = 0.0087). The fluorescence intensity of the whole ischemic brain was correlated with the NaFL concentration of brain tissue homogenates (r = 0.8653, P = 0.0026) and cerebral infarction volume (r = 0.7282, P = 0.0072). Comparison with existing methodsThe new method enables concurrent measurement of BBB permeability and infarction volume on the same batch of brain tissue samples without affecting most downstream biochemical assays. ConclusionsBy applying the new method, we could use the same batch of ischemic rodent brain tissue for multiple assays, including BBB permeability and infarction volume. Through this, we would reduce the animal numbers in each study and help to maximize the scientific and statistical potential of future rodent ischemic studies.

Orexin 2 receptor (OX2R) protein distribution measured by autoradiography using radiolabeled OX2R-selective antagonist EMPA in rodent brain and peripheral tissues

Orexin, a neuropeptide, performs various physiological functions, including the regulation of emotion, feeding, metabolism, respiration, and sleep/wakefulness, by activating the orexin 1 receptor and orexin 2 receptor (OX2R). Owing to the pivotal role of OX2R in wakefulness and other biological functions, OX2R agonists are being developed. A detailed understanding of OX2R protein distribution is essential for determining the mechanisms of action of OX2R agonists; however, this has been hindered by the lack of selective antibodies. In this study, we first confirmed the OX2R-selective binding of [3H]-EMPA in in vitro autoradiography studies, using brain slices from OX2R knockout mice and their wild-type littermates. Subsequently, OX2R protein distribution in rats was comprehensively assessed in 51 brain regions and 10 peripheral tissues using in vitro autoradiography with [3H]-EMPA. The widespread distribution of OX2R protein, including that in previously unrecognized regions of the retrosplenial cortex, was identified. In contrast, OX2R protein expression was negligible/very low in peripheral tissues, suggesting that orexin exerts OX2R-dependent physiological functions primarily through activation of the central nervous system. These findings will be useful for understanding the wide range of biological functions of OX2R and the application of OX2R agonists in various disorders.

Implementation of Minimally Invasive Brain Tumor Resection in Rodents for High Viability Tissue Collection.

The present protocol describes a standardized paradigm for rodent brain tumor resection and tissue preservation. In clinical practice, maximal tumor resection is the standard-of-care treatment for most brain tumors. However, most currently available preclinical brain tumor models either do not include resection, or utilize surgical resection models that are time-consuming and lead to significant postoperative morbidity, mortality, or experimental variability. In addition, performing resection in rodents can be daunting for several reasons, including a lack of clinically comparable surgical tools or protocols and the absence of an established platform for standardized tissue collection. This protocol highlights the use of a multi-functional, non-ablative resection device and an integrated tissue preservation system adapted from the clinical version of the device. The device applied in the present study combines tunable suction and a cylindrical blade at the aperture to precisely probe, cut, and suction tissue. The minimally invasive resection device performs its functions via the same burr hole used for the initial tumor implantation. This approach minimizes alterations to regional anatomy during biopsy or resection surgeries and reduces the risk of significant blood loss. These factors significantly reduced the operative time (<2 min/animal), improved postoperative animal survival, lower variability in experimental groups, and result in high viability of resected tissues and cells for future analyses. This process is facilitated by a blade speed of ~1,400 cycles/min, which allows the harvesting of tissues into a sterile closed system that can be filled with a physiologic solution of choice. Given the emerging importance of studying and accurately modeling the impact of surgery, preservation and rigorous comparative analysis of regionalized tumor resection specimens, and intra-cavity-delivered therapeutics, this unique protocol will expand opportunities to explore unanswered questions about perioperative management and therapeutic discovery for brain tumor patients.

Activation of lactate receptor HCAR1 down-modulates neuronal activity in rodent and human brain tissue

Lactate can be used by neurons as an energy substrate to support their activity. Evidence suggests that lactate also acts on a metabotropic receptor called HCAR1, first described in the adipose tissue. Whether HCAR1 also modulates neuronal circuits remains unclear. In this study, using qRT-PCR, we show that HCAR1 is present in the human brain of epileptic patients who underwent resective surgery. In brain slices from these patients, pharmacological HCAR1 activation using a non-metabolized agonist decreased the frequency of both spontaneous neuronal Ca2+ spiking and excitatory post-synaptic currents (sEPSCs). In mouse brains, we found HCAR1 expression in different regions using a fluorescent reporter mouse line and in situ hybridization. In the dentate gyrus, HCAR1 is mainly present in mossy cells, key players in the hippocampal excitatory circuitry and known to be involved in temporal lobe epilepsy. By using whole-cell patch clamp recordings in mouse and rat slices, we found that HCAR1 activation causes a decrease in excitability, sEPSCs, and miniature EPSCs frequency of granule cells, the main output of mossy cells. Overall, we propose that lactate can be considered a neuromodulator decreasing synaptic activity in human and rodent brains, which makes HCAR1 an attractive target for the treatment of epilepsy.

Brain virtual histology with X-ray phase-contrast tomography Part I: whole-brain myelin mapping in white-matter injury models.

White-matter injury leads to severe functional loss in many neurological diseases. Myelin staining on histological samples is the most common technique to investigate white-matter fibers. However, tissue processing and sectioning may affect the reliability of 3D volumetric assessments. The purpose of this study was to propose an approach that enables myelin fibers to be mapped in the whole rodent brain with microscopic resolution and without the need for strenuous staining. With this aim, we coupled in-line (propagation-based) X-ray phase-contrast tomography (XPCT) to ethanol-induced brain sample dehydration. We here provide the proof-of-concept that this approach enhances myelinated axons in rodent and human brain tissue. In addition, we demonstrated that white-matter injuries could be detected and quantified with this approach, using three animal models: ischemic stroke, premature birth and multiple sclerosis. Furthermore, in analogy to diffusion tensor imaging (DTI), we retrieved fiber directions and DTI-like diffusion metrics from our XPCT data to quantitatively characterize white-matter microstructure. Finally, we showed that this non-destructive approach was compatible with subsequent complementary brain sample analysis by conventional histology. In-line XPCT might thus become a novel gold-standard for investigating white-matter injury in the intact brain. This is Part I of a series of two articles reporting the value of in-line XPCT for virtual histology of the brain; Part II shows how in-line XPCT enables the whole-brain 3D morphometric analysis of amyloid- (A ) plaques.

Rodent Heart and Brain Tissue Preparation for Digital Macro Photography after Ischemia-reperfusion

Macro photography is applicable for imaging various tissue samples at high magnification to perform qualitative and quantitative analyses. Tissue preparation and subsequent image capture are steps performed immediately after the ischemia-reperfusion (IR) experiment and must be performed in a timely manner and with appropriate care. For the evaluation of IR-induced damage in the heart and brain, this paper describes 2,3,5-triphenyl-2H-tetrazolium chloride (TTC)-based staining followed by macro photography. Scientific macro photography requires controlled lighting and an appropriate imaging setup. The standardized methodology ensures high-quality, detailed digital images even if a combination of an inexpensive up-to-date digital camera and macro lens is used. Proper techniques and potential mistakes in sample preparation and image acquisition are discussed, and examples of the influence of correct and incorrect setups on image quality are provided. Specific tips are provided on how to avoid common mistakes, such as overstaining, improper sample storage, and suboptimal lighting conditions. This paper shows the appropriate methodology for rat heart and brain tissue slicing and staining and provides guidelines for establishing lighting and camera setups and photography techniques for high-resolution image acquisition.

Rodent Heart and Brain Tissue Preparation for Digital Macro Photography after Ischemia-reperfusion.

Macro photography is applicable for imaging various tissue samples at high magnification to perform qualitative and quantitative analyses. Tissue preparation and subsequent image capture are steps performed immediately after the ischemia-reperfusion (IR) experiment and must be performed in a timely manner and with appropriate care. For the evaluation of IR-induced damage in the heart and brain, this paper describes 2,3,5-triphenyl-2H-tetrazolium chloride (TTC)-based staining followed by macro photography. Scientific macro photography requires controlled lighting and an appropriate imaging setup. The standardized methodology ensures high-quality, detailed digital images even if a combination of an inexpensive up-to-date digital camera and macro lens is used. Proper techniques and potential mistakes in sample preparation and image acquisition are discussed, and examples of the influence of correct and incorrect setups on image quality are provided. Specific tips are provided on how to avoid common mistakes, such as overstaining, improper sample storage, and suboptimal lighting conditions. This paper shows the appropriate methodology for rat heart and brain tissue slicing and staining and provides guidelines for establishing lighting and camera setups and photography techniques for high-resolution image acquisition.

Uncertain histological origin of "glia-like" cells in adult human brain tissue cultures.

Most brain cells studies come from cultured rodent brain tissue, so basic questions about the behaviour of cultured adult human glial cells may remain unanswered. Cells cultured from adult human brain have been poorly defined until now and are often termed "glia-like" based on some morphological similarities with astrocytes. However, the cells in question fail to express glial markers and may be well be of non-glial origin. We examined adult human brain and cultures from 10 patients with non-malignant diagnoses. Immunofluorescence methods were used for glial and non-glial cell type identifications. Confluent cultures contained the following: 0.1 % astrocytes, ≤ 0.01 % oligodendrocytes, 2-5 % microglial and 95-98 % "glia-like" cells. Astrocytes tested as followed: GFAP+/Vim+, microglia: Ferr+Vim+, "glia-like" cells: Vim+/Fn+/CK- or CK+. In the brain tissue, astrocytes were GFAP+/Vim+, microglia Ferr+/Vim-, fibronectin expression was restricted to brain vessels. This report demonstrates considerable morphological and cytoskelatal dedifferentiation of cultured brain cells. Cytokeratins, specific markers for epithelial cell differentiation, were absent in the brain tissue. However, they were expressed in "glia-like" cells. This finding could be considered glial dedifferentiation given the ectodermal origin of the brain tissue. We suggest that "glia-like" cells come from currently unknown glial progenitor cells scattered through the brain tissue (Tab. 1, Fig. 4, Ref. 19).

The Neuroprotective Effects of Cannabis-Derived Phytocannabinoids and Resveratrol in Parkinson's Disease: A Systematic Literature Review of Pre-Clinical Studies.

Currently, there are no pharmacological treatments able to reverse nigral degeneration in Parkinson’s disease (PD), hence the unmet need for the provision of neuroprotective agents. Cannabis-derived phytocannabinoids (CDCs) and resveratrol (RSV) may be useful neuroprotective agents for PD due to their anti-oxidative and anti-inflammatory properties. To evaluate this, we undertook a systematic review of the scientific literature to assess the neuroprotective effects of CDCs and RSV treatments in pre-clinical in vivo animal models of PD. The literature databases MEDLINE, EMBASE, PsychINFO, PubMed, and Web of Science core collection were systematically searched to cover relevant studies. A total of 1034 publications were analyzed, of which 18 met the eligibility criteria for this review. Collectively, the majority of PD rodent studies demonstrated that treatment with CDCs or RSV produced a significant improvement in motor function and mitigated the loss of dopaminergic neurons. Biochemical analysis of rodent brain tissue suggested that neuroprotection was mediated by anti-oxidative, anti-inflammatory, and anti-apoptotic mechanisms. This review highlights the neuroprotective potential of CDCs and RSV for in vivo models of PD and therefore suggests their potential translation to human clinical trials to either ameliorate PD progression and/or be implemented as a prophylactic means to reduce the risk of development of PD.

Nonlinearity of the coefficient of thermal expansion in brain tissue

The coefficient of thermal expansion (CTE) in biological tissues is an integral parameter behind the application of electromagnetic energy to biomedical technologies; however, its behavior is far from being fully characterized. In this study, we apply digital image correlation (DIC) to non-invasively measure the microscale thermal expansions of recently excised embryonic E18 rodent brain tissue slices. Although the CTE has been measured previously in soft tissues, the literature surrounding the expansion of brain tissue remains sparse. Previous work in measuring the thermal expansion behavior of soft tissue often simplifies the results into a single measurement of a linear CTE parameter and fails to convey the temperature-dependent nonlinearity that exists. In this work, we demonstrate that: (1) the coefficient of brain tissue is more similar to fat than blood, and (2) there exists a significant nonlinear increase in CTE at physiologically-relevant temperatures. This suggests some limitations with the interpretation of previously reported values of the CTE, which are often measured at room temperature.

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues

We report a simple, time-efficient and high-throughput fluorescence spectroscopy-based assay for the quantification of actin filaments in ex vivo biological samples from brain tissues of rodents and human subjects.

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues.

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin occurs in equilibrium in its two forms: monomeric globular (G-actin) and polymerized filamentous (F- actin). At the synaptic terminals, actin cytoskeleton forms the basis for critical pre- and post-synaptic functions. Moreover, dynamic changes in the actin polymerization status (interconversion between globular and filamentous forms of actin) are closely linked to plasticity-related alterations in synaptic structure and function. We report here a modified fluorescence-based methodology to assess polymerization status of actin in ex vivo conditions. The assay employs fluorescently labelled phalloidin, a phallotoxin that specifically binds to actin filaments (F-actin), providing a direct measure of polymerized filamentous actin. As a proof of principle, we provide evidence for the suitability of the assay both in rodent and post-mortem human brain tissue homogenates. Using latrunculin A (a drug that depolymerizes actin filaments), we confirm the utility of the assay in monitoring alterations in F-actin levels. Further, we extend the assay to biochemical fractions of isolated synaptic terminals wherein we confirm increased actin polymerization upon stimulation by depolarization with high extracellular K+.

Chemical Stimulation of Rodent and Human Cortical Synaptosomes: Implications in Neurodegeneration.

Synaptic plasticity events, including long-term potentiation (LTP), are often regarded as correlates of brain functions of memory and cognition. One of the central players in these plasticity-related phenomena is the α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor (AMPAR). Increased levels of AMPARs on postsynaptic membranes thus constitute a biochemical measure of LTP. Isolated synaptic terminals (synaptosomes) are an excellent ex vivo tool to monitor synaptic physiology in healthy and diseased brains, particularly in human research. We herein describe three protocols for chemically-induced LTP (cLTP) in synaptosomes from both rodent and human brain tissues. Two of these chemical stimulation protocols are described for the first time in synaptosomes. A pharmacological block of synaptosomal actin dynamics confirmed the efficiency of the cLTP protocols. Furthermore, the study prototypically evaluated the deficiency of cLTP in cortical synaptosomes obtained from human cases of early-onset Alzheimer’s disease (EOAD) and frontotemporal lobar degeneration (FLTD), as well as an animal model that mimics FLTD.

Anti-NMDA Receptor Encephalitis

Anti-NMDA receptor (NMDAR) encephalitis is an autoimmune disease caused by autoantibodies against the extracellular conformational epitope of the NR1 subunit of the NMDAR (GluN1 antibodies). A series of autoantibodies directed against neuronal surface (NS) or synaptic proteins play an important role in the pathophysiological mechanisms of post-herpes simplex encephalitis (post-HSE), overlapping autoimmune encephalitis and demyelinating syndrome, epileptic seizures, psychosis, involuntary movements (orofacial and limb dyskinesias, catatonia, dystonia, chorea, myoclonus, psychogenic nonepileptic seizures, and faciobrachial dystonic seizures), postpartum psychosis, stiff-person spectrum disorder (including progressive encephalomyelitis with rigidity and myoclonus [PERM]), cerebellar ataxia, and sleep behavior disorders. These NS antibodies are identified with cell-based assays and immunohistochemistry using nonperfused paraformaldehyde-fixed rodent brain tissue. This paper presents an update on anti-NMDAR encephalitis, including the differential diagnosis of cryptogenic new-onset refractory status epilepticus (NORSE), and on the treatment strategy, including third-line therapy.

Cuprizone markedly decreases kynurenic acid levels in the rodent brain tissue and plasma

BackgroundThe kynurenine (KYN) pathway (KP) of the tryptophan (TRP) metabolism seems to play a role in the pathomechanism of multiple sclerosis (MS). Cuprizone (CPZ) treated animals develop both demyelination (DEM) and remyelination (REM) in lack of peripheral immune response, such as the lesion pattern type III and IV in MS, representing primary oligodendrogliopathy. ObjectiveTo measure the metabolites of the KP in the CPZ treated animals, including TRP, KYN and kynurenic acid (KYNA). We proposed that KYNA levels might be decreased in the CPZ-induced demyelinating phase of the animal model of MS, which model represents the progressive phase of the disease. MethodsA total of 64 C57Bl/6J animals were used for the study. Immunohistochemical (IHC) measurements were performed to prove the effect of CPZ, whereas high-performance liquid chromatography (HPLC) was used to quantify the metabolites of the KP (n = 10/4 groups; DEM, CO1, REM, CO2). ResultsIHC measurements proved the detrimental effects of CPZ. HPLC measurements demonstrated a decrease of KYNA in the hippocampus (p < 0.05), somatosensory cortex (p < 0.01) and in plasma (p < 0.001). ConclusionThis is the first evidence of marked reduction in KYNA levels in a non-immune mediated model of MS. Our results suggest an involvement of the KP in the pathomechanism of MS, which needs to be further elucidated.

Isolation and Propagation of Primary Human and Rodent Embryonic Neural Progenitor Cells and Cortical Neurons.

The research on human neural progenitor cells holds great potential for the understanding of the molecular programs that control differentiation of cells of glial and neuronal lineages, as well as pathogenetic mechanisms of neurological diseases. Stem cell technologies also provide opportunities for the pharmaceutical industry to develop new approaches for regenerative medicine. Here, we describe the protocol for the isolation and maintenance of neural progenitor cells and cortical neurons using human fetal brain tissue. This protocol can be successfully adapted for the preparation of rodent neural and oligodendrocyte progenitor cells. While several methods for isolating neural and oligodendrocyte progenitors from rodent brain tissue have been described, including techniques utilizing gene transfer and magnetic resonance beads, few methods are specifically focused on deriving human oligodendrocyte progenitor cells. Development of the human cultures provides the most physiologically relevant system for investigating mechanisms which regulate the function of oligodendrocytes, specifically of human origin.

Investigating protein expression, modifications and interactions in the brain: Protocol for preparing rodent brain tissue for mass spectrometry-based quantitative- and phospho-proteomics analysis.

Neurodegenerative diseases remain a major global health challenge, affecting millions of individuals worldwide. Despite significant research efforts, the etiology and pathological mechanisms of most neurodegenerative disorders remain uncertain. This indicates that new disease models and research strategies need to be developed to enhance biomarker discovery and drug development processes. Studies have shown that dysregulation of protein-protein interactions in the brain may be key to the pathophysiological processes in a number of neurodegenerative diseases. Consequently, a deeper understanding of protein-protein interactions in the brain in both healthy and neurodegenerative states may provide greater insights into disease mechanisms and progress, and aid the development of preventive, management and treatment strategies. Efforts to investigate protein-protein interactions in the brain have largely depended on purifying the protein, together with its interacting partner proteins (interactome) and then analyzing these interactions by mass spectrometry. However, factors including abundance and phosphorylation status of the protein of interest and its interactome can lead to identification of false protein-protein interactions. Moreover, studies have shown that combining quantitative proteomics data with data from affinity-purification-mass spectrometry data can reduce these false positives and also provide more insights into protein-protein interactions. Thus, we have developed a protocol for preparing rodent brain tissue for quantitative- and phospho-proteomics analysis, combining in-house and commercially available kits. Data from this protocol will enhance the analysis and interpretation of protein-protein interactions in both physiological and pathological states.

Quantification of SV2A Binding in Rodent Brain Using [18F]SynVesT-1 and PET Imaging.

Synapse loss is a hallmark of Alzheimer's disease (AD) and correlates with cognitive decline. The validation of a noninvasive in vivo imaging approach to quantify synapse would greatly facilitate our understanding of AD pathogenesis and assist drug developments for AD. As animal models of neurodegenerative and neuropsychiatric disorders play a critical role in the drug discovery and development process, a robust, objective, and translational method for quantifying therapeutic drug efficacy in animal models will facilitate the drug development process. In this study, we tested the quantification reliability of the SV2A PET tracer, [18F]SynVesT-1, in a mouse model of AD (APP/PS1) and wild-type controls, and developed a simplified quantification method to facilitate large cohort preclinical imaging studies. We generated nondisplaceable binding potential (BPND) and distribution volume ratio (DVR) values using the simplified reference tissue model (SRTM) on the 90-min dynamic PET imaging data, with brain stem and cerebellum as the reference region, respectively. Then, we correlated the standardized uptake value ratio (SUVR)-1 and SUVR averaged from different imaging windows with BPND and DVR, using brain stem and cerebellum as the reference region, respectively. We performed homologous competitive binding assay and autoradiographic saturation binding assay using [18F]SynVesT-1 to calculate the Bmax and Kd. Using brain stem as the reference region, the averaged SUVR-1 from 30 to 60 min postinjection correlated well with the BPND calculated using SRTM. Using cerebellum as the reference region, the averaged SUVR from 30 to 60 min postinjection correlated well with the SRTM DVR. From the homologous competitive binding assay and autoradiographic saturation binding assay, the calculated the Bmax and Kd were 4.5-18 pmol/mg protein and 9.8-19.6 nM, respectively, for rodent brain tissue. This simplified SUVR method provides reasonable SV2A measures in APP/PS1 mice and their littermate controls. Our data indicate that, in lieu of a full 90-min dynamic scan, a 30-min static PET scan (from 30 to 60 min postinjection) would be sufficient to provide quantification data on SV2A expression, equivalent to the data generated from kinetic modeling. The methods developed here are readily applicable to the evaluation of therapeutic effects of novel drugs in this rodent model using [18F]SynVesT-1 and small animal PET.

Depth-resolved imaging of photosensitizer in the rodent brain using fluorescence laminar optical tomography.

.Significance: Previous studies have been performed to image photosensitizers in certain organs and tumors using fluorescence laminar optical tomography. Currently, no work has yet been published to quantitatively compare the signal compensation of fluorescence laminar optical tomography with two-dimensional (2-D) imaging in tissues.Aim: The purpose of this study is to quantify the benefit that fluorescence laminar optical tomography holds over 2-D imaging. We compared fluorescence laminar optical tomography with maximum intensity projection imaging to simulate 2-D imaging, as this would be the most similar and stringent comparison.Approach: A capillary filled with a photosensitizer was placed in a phantom and ex vivo rodent brains, with fluorescence laminar optical tomography and maximum intensity projection images obtained. The signal loss in the direction was quantified and compared to see which methodology could compensate better for signal loss caused by tissue attenuation.Results: The results demonstrated that we can reconstruct a capillary filled with benzoporphyrin derivative photosensitizers faithfully in phantoms and in ex vivo rodent brain tissues using fluorescence laminar optical tomography. We further demonstrated that we can better compensate for signal loss when compared with maximum intensity projection imaging.Conclusions: Using fluorescence laminar optical tomography (FLOT), one can compensate for signal loss in deeper parts of tissue when imaging in ex vivo rodent brain tissue compared with maximum intensity projection imaging.

Modification of methods to use Congo-red stain to simultaneously visualize amyloid plaques and tangles in human and rodent brain tissue sections.

Although there are multiple histochemical tracers available to label plaques and tangles in the brain to evaluate neuropathology in Alzheimer disease (AD), few of them are versatile in nature and compatible with immunohistochemical procedures. Congo Red (CR) is an anisotropic organic stain discovered to label amyloid beta (Aβ) plaques in the brain. Unfortunately, its use is underappreciated due to its low resolution and brightness as stated in previous studies using bright field microscopy. Here, we modified a previous method to localize both plaques and tangles in brains from humans and a transgenic rodent model of AD for fluorescence microscopic visualization. The plaque staining affinities displayed by CR were compared with fibrillar pattern labeling seen with Thioflavin S. This study summarizes the optimization of protocols in which various parameters have been finetuned. To determine the target CR potentially binds, we have performed double labeling with different antibodies against Aβ as well as phosphorylated Tau. The plaque staining affinities exhibited by CR are compared with those associated with the diffuse pattern of labeling seen with antibodies directed against different epitopes of Aβ. Neither CP13, TNT2 or TOC1 binds all the neurofibrillary tangles as revealed by CR labeling in the human brain. Additionally, we also evaluated double labeling with AT8, AT180, and PHF1. Interestingly, PHF-1 shows 40% colocalization and AT8 shows 15% colocalization with NFT. Thus, CR is a much better marker to detect AD pathologies in human and rodent brains with higher fluorescence intensity relative to other conventional fluorescence markers.