Iba1 immunoreactivity is enhanced following an antigen retrieval treatment with EDTA, pH 6.0

Ghatak, Atreyi; Combs, Colin K.

doi:10.1016/j.mex.2014.10.007

Cited by 12 publications

(8 citation statements)

References 3 publications

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“…Technical differences could also contribute to variation between studies. For immunohistochemistry, the most commonly used technique in this review, the method and duration of tissue fixation, 188 , 189 the thickness of the brain sections, 189 the use of antigen retrieval, 190 the antibody selected 77 , 188 and variation in many steps in the immunohistochemistry protocol itself can all influence the intensity of staining that could affect the ability of studies to pick up differences between AD and control subjects. Differences in the methods used for measuring gene expression or for quantifying protein by western blot or enzyme-linked immunosorbent assay could similarly contribute to variability in the results.…”

Section: Discussionmentioning

confidence: 99%

Markers of microglia in post-mortem brain samples from patients with Alzheimer’s disease: a systematic review

et al. 2017

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Neuroinflammation is proposed as one of the mechanisms by which Alzheimer’s disease pathology, including amyloid-β plaques, leads to neuronal death and dysfunction. Increases in the expression of markers of microglia, the main neuroinmmune cell, are widely reported in brains from patients with Alzheimer’s disease, but the literature has not yet been systematically reviewed to determine whether this is a consistent pathological feature. A systematic search was conducted in Medline, Embase and PsychINFO for articles published up to 23 February 2017. Papers were included if they quantitatively compared microglia markers in post-mortem brain samples from patients with Alzheimer’s disease and aged controls without neurological disease. A total of 113 relevant articles were identified. Consistent increases in markers related to activation, such as major histocompatibility complex II (36/43 studies) and cluster of differentiation 68 (17/21 studies), were identified relative to nonneurological aged controls, whereas other common markers that stain both resting and activated microglia, such as ionized calcium-binding adaptor molecule 1 (10/20 studies) and cluster of differentiation 11b (2/5 studies), were not consistently elevated. Studies of ionized calcium-binding adaptor molecule 1 that used cell counts almost uniformly identified no difference relative to control, indicating that increases in activation occurred without an expansion of the total number of microglia. White matter and cerebellum appeared to be more resistant to these increases than other brain regions. Nine studies were identified that included high pathology controls, patients who remained free of dementia despite Alzheimer’s disease pathology. The majority (5/9) of these studies reported higher levels of microglial markers in Alzheimer’s disease relative to controls, suggesting that these increases are not solely a consequence of Alzheimer’s disease pathology. These results show that increased markers of microglia are a consistent feature of Alzheimer’s disease, though this seems to be driven primarily by increases in activation-associated markers, as opposed to markers of all microglia.

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Section: Discussionmentioning

confidence: 99%

Markers of microglia in post-mortem brain samples from patients with Alzheimer’s disease: a systematic review

et al. 2017

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“…Serial, 8 μm thick, slices were cut, mounting every 5th section on Leica X-tra TM Adhesive Micro slides (SMM Instruments, South Africa). Antigen retrieval was performed following the EDTA, pH 6.0 protocol by Ghatak and Combs (2014). Briefly, mounted slides were placed inside a glass coplin jar with EDTA, pH 6.0, after which the sealed coplin jar was boiled for 10 min in a 500 mL beaker filled with water.…”

Section: Methodsmentioning

confidence: 99%

Oleanolic Acid Mitigates 6-Hydroxydopamine Neurotoxicity by Attenuating Intracellular ROS in PC12 Cells and Striatal Microglial Activation in Rat Brains

Msibi

Mabandla

2019

Front. Physiol.

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Oleanolic acid (OA), a biologically active pentacyclic triterpenoid compound, has been implicated in a number of clinical benefits including antioxidant, and anti-inflammatory properties. OA has been previously shown to ameliorate the toxic effects of 6-hydroxydopamine (6-OHDA), however, the mechanism by which this effect is exhibited is not clearly understood. In the present study, we investigated the role of OA in attenuation of microglial activation in 6-OHDA induced Parkinsonian rat model. We also explored the ability of OA to attenuate 6-OHDA-induced intracellular reactive oxygen species (ROS), and thus prevent cell death in PC12 cells. We accessed the utility of immunohistochemistry to assess striatal microglial activation, where shape descriptors such as area, perimeter, Feret’s diameter, aspect ratio and solidity were determined using the Fiji ImageJ software. Intracellular ROS and cell viability were assessed in PC12 cells using the OxiSelect TM Intracellular ROS Assay Kit and MTT assay, respectively. We found that microglial activation was decreased in rats pre-treated with OA prior to 6-OHDA insult as well as in rats treated with OA 1 day post 6-OHDA exposure when compared to untreated rats, as determined by shape descriptors. This finding was in correlation with significantly improved motor symptoms and increased striatal dopamine in treated rats as compared to non-treated rats. Flow cytometry assessment of PC12 cells revealed a decreased amount of intracellular ROS in cells pre-treated with OA 6 h prior to 6-OHDA exposure and cells treated with OA 1 h post 6-OHDA exposure, suggesting that OA provides neuroprotection in PC12 cells by removing intracellular ROS, thereby reducing oxidative stress. Our finding suggest that OA exhibits its neuroprotective effect by attenuating striatal microglial activation, which results in neuroinflammation that is implicated in Parkinson’s disease pathology. Further studies detailing the mechanism by which OA interacts with microglia may be useful in understanding the role of OA in attenuating neuroinflammation.

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“…Brain sections were mounted on glass slide. Once dried, brain sections underwent to a heat-induced antigen retrieval step in a 10 mM EDTA solution, pH = 6.0, for 20 min at 55°C [51]. After 4 washes with filtered PBS, sections were blocked with a solution containing 10% normal goat serum and 0.2% Triton X-100 in filtered PBS.…”

Section: Immunofluorescencementioning

confidence: 99%

The Dysregulation of OGT/OGA Cycle Mediates Tau and APP Neuropathology in Down Syndrome

et al. 2021

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Protein O-GlcNAcylation is a nutrient-related post-translational modification that, since its discovery some 30 years ago, has been associated with the development of neurodegenerative diseases. As reported in Alzheimer’s disease (AD), flaws in the cerebral glucose uptake translate into reduced hexosamine biosynthetic pathway flux and subsequently lead to aberrant protein O-GlcNAcylation. Notably, the reduction of O-GlcNAcylated proteins involves also tau and APP, thus promoting their aberrant phosphorylation in AD brain and the onset of AD pathological markers. Down syndrome (DS) individuals are characterized by the early development of AD by the age of 60 and, although the two conditions present the same pathological hallmarks and share the alteration of many molecular mechanisms driving brain degeneration, no evidence has been sought on the implication of O-GlcNAcylation in DS pathology. Our study aimed to unravel for the first time the role of protein O-GlcNacylation in DS brain alterations positing the attention of potential trisomy-related mechanisms triggering the aberrant regulation of OGT/OGA cycle. We demonstrate the disruption of O-GlcNAcylation homeostasis, as an effect of altered OGT and OGA regulatory mechanism, and confirm the relevance of O-GlcNAcylation in the appearance of AD hallmarks in the brain of a murine model of DS. Furthermore, we provide evidence for the neuroprotective effects of brain-targeted OGA inhibition. Indeed, the rescue of OGA activity was able to restore protein O-GlcNAcylation, and reduce AD-related hallmarks and decreased protein nitration, possibly as effect of induced autophagy.

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Iba1 immunoreactivity is enhanced following an antigen retrieval treatment with EDTA, pH 6.0

Cited by 12 publications

References 3 publications

Markers of microglia in post-mortem brain samples from patients with Alzheimer’s disease: a systematic review

Markers of microglia in post-mortem brain samples from patients with Alzheimer’s disease: a systematic review

Oleanolic Acid Mitigates 6-Hydroxydopamine Neurotoxicity by Attenuating Intracellular ROS in PC12 Cells and Striatal Microglial Activation in Rat Brains

The Dysregulation of OGT/OGA Cycle Mediates Tau and APP Neuropathology in Down Syndrome

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