A Low Cost Antibody Signal Enhancer Improves Immunolabeling in Cell Culture, Primate Brain and Human Cancer Biopsy

Flores-Maldonado, Catalina; Albino‑Sanchez, M. E.; Rodríguez-Callejas, Juan D.; Estrada-Mondragón, Argel; León-Galicia, Ismael; Pérez-Cruz, Claudia; Fuchs, Eberhard; García‐Carrancá, Alejandro; Contreras, Rubén G.; Missirlis, Fanis; Rosas‐Arellano, Abraham

doi:10.1016/j.neuroscience.2020.01.009

Cited by 13 publications

(8 citation statements)

References 62 publications

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“…Fixed sperm were permeabilized in 0.2% Triton X-100 at room temperature for 10 min. Then, the sperm were washed in PBST and then the coverslips were blocked in Antigen Signal Enhancer (ASE) blocking solution (50 mM glycine, 0.05% Tween 20, 0.1% Triton X-100, and 10% NGS in PBS) [ 73 ] for 30 min at room temperature. The coverslips containing the fixed sperm were incubated overnight at 4 °C in ASE blocking solution containing 20 µg/mL of our anti-NHE11 antibody.…”

Section: Methodsmentioning

confidence: 99%

The SLC9C2 Gene Product (Na+/H+ Exchanger Isoform 11; NHE11) Is a Testis-Specific Protein Localized to the Head of Mature Mammalian Sperm

Gardner

James

2023

IJMS

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Na+/H+ exchangers (NHEs) are a family of ion transporters that regulate the pH of various cell compartments across an array of cell types. In eukaryotes, NHEs are encoded by the SLC9 gene family comprising 13 genes. SLC9C2, which encodes the NHE11 protein, is the only one of the SLC9 genes that is essentially uncharacterized. Here, we show that SLC9C2 exhibits testis/sperm-restricted expression in rats and humans, akin to its paralog SLC9C1 (NHE10). Similar to NHE10, NHE11 is predicted to contain an NHE domain, a voltage sensing domain, and finally an intracellular cyclic nucleotide binding domain. An immunofluorescence analysis of testis sections reveals that NHE11 localizes with developing acrosomal granules in spermiogenic cells in both rat and human testes. Most interestingly, NHE11 localizes to the sperm head, likely the plasma membrane overlaying the acrosome, in mature sperm from rats and humans. Therefore, NHE11 is the only known NHE to localize to the acrosomal region of the head in mature sperm cells. The physiological role of NHE11 has yet to be demonstrated but its predicted functional domains and unique localization suggests that it could modulate intracellular pH of the sperm head in response to changes in membrane potential and cyclic nucleotide concentrations that are a result of sperm capacitation events. If NHE11 is shown to be important for male fertility, it will be an attractive target for male contraceptive drugs due to its exclusive testis/sperm-specific expression.

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Section: Methodsmentioning

confidence: 99%

The SLC9C2 Gene Product (Na+/H+ Exchanger Isoform 11; NHE11) Is a Testis-Specific Protein Localized to the Head of Mature Mammalian Sperm

Gardner

James

2023

IJMS

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“…To block potential nonspecific antibody binding, sections were incubated for 30 min using a solution containing 2 % donkey serum, 50 mM glycine, 0.05 % Tween20, 0.1 % TritonX-100, and 0.1 % BSA diluted in PBS. Primary antibody anti-GFAP (1:300, goat/IgG, Abcam, Ab53554) was incubated in an antibody signal enhancer (ASE) solution (Flores-Maldonado et al, 2020; Rosas-Arellano et al, 2016), consisting of 10 mM glycine, 0.05 % Tween20, 0.1 % TritonX-100 and 0.1 % hydrogen peroxide in PBS, and left overnight at 4 °C. For double labeling, GFAP antibody was incubated with 8-hydroxyguanosine (8OHG, marker of RNA oxidation) (1:10000, mouse/IgG, Abcam, ab62623), AT100 (1:500, mouse/IgG, Thermo Scientific, MN1060), and S100A10 (1:250, mouse/IgG, Invitrogen, PIMA515326) primary antibodies.…”

Section: Methodsmentioning

confidence: 99%

“…The quantification of GFAP fluorescence intensity was performed in 1440 astrocytes. This analysis was performed according to previous studies (Flores-Maldonado et al, 2020; Rosas-Arellano et al, 2016). Using the ImageJ “free hand” function the cytoplasm and processes of the astrocytes were outlined.…”

Section: Methodsmentioning

confidence: 99%

“…Primary antibody anti-GFAP (1:300, goat/IgG, Abcam, Ab53554) was incubated in an antibody signal enhancer (ASE) solution (Flores-Maldonado et al, 2020;Rosas-Arellano et al, 2016), consisting of 10 mM glycine, 0.05 % Tween20, 0.1 % TritonX-100 and 0.1 % hydrogen peroxide in PBS, and left overnight at 4 °C. For double labeling, GFAP antibody was incubated with 8-hydroxyguanosine (8OHG, marker of RNA oxidation)…”

Section: Double Labeling Immunofluorescencementioning

confidence: 99%

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Atrophic astrocytes in aged marmosets present tau hyperphosphorylation, RNA oxidation, and DNA fragmentation

Rodríguez-Callejas¹,

Fuchs²,

Pérez-Cruz³

2023

Preprint

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Astrocytes perform multiple essential functions in the brain showing morphological changes. Hypertrophic astrocytes are commonly observed in cognitively healthy aged animals, implying a functional defense mechanism without losing neuronal support. In neurodegenerative diseases, astrocytes show morphological alterations, such as decreased process length and reduced number of branch points, known as astroglial atrophy, with detrimental effects on neuronal cells. The common marmoset (Callithrix jacchus) is a non-human primate that, with age, develops several features that resemble neurodegeneration. In this study, we characterize the morphological alterations in astrocytes of adolescent (mean 1.75 y), adult (mean 5.33 y), old (mean 11.25 y), and aged (mean 16.83 y) male marmosets. We observed a significantly reduced arborization in astrocytes of aged marmosets compared to younger animals in the hippocampus and entorhinal cortex. These astrocytes also show oxidative damage to RNA and increased nuclear pTau (AT100). Astrocytes lacking S100A10 protein show a more severe atrophy and DNA fragmentation. Our results demonstrate the presence of atrophic astrocytes in the brains of aged marmosets.

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“…Before staining, sections were washed in PBS for 2 times for 10 min each. They were blocked in an antibody signal enhancer blocking buffer 43,44 then stained either overnight or over the weekend with EnCor rabbit anti‐c‐Fos (EnCor Biotechnology cat no. RPCA‐c‐FOS‐AP, ) at a 1 to 5000 dilution in ASE primary buffer, floating.…”

Section: Methodsmentioning

confidence: 99%

Constitutive and conditional deletion reveals distinct phenotypes driven by developmental versus neurotransmitter actions of the neuropeptide PACAP

Bakalar

Гаврилова

Jiang

et al. 2023

J Neuroendocrinology

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Neuropeptides may exert trophic effects during development, and then neurotransmitter roles in the developed nervous system. One way to associate peptide‐deficiency phenotypes with either role is first to assess potential phenotypes in so‐called constitutive knockout mice, and then proceed to specify, regionally and temporally, where and when neuropeptide expression is required to prevent these phenotypes. We have previously demonstrated that the well‐known constellation of behavioral and metabolic phenotypes associated with constitutive pituitary adenylate cyclase‐activating peptide (PACAP) knockout mice are accompanied by transcriptomic alterations of two types: those that distinguish the PACAP‐null phenotype from wild‐type (WT) in otherwise quiescent mice (cPRGs), and gene induction that occurs in response to acute environmental perturbation in WT mice that do not occur in knockout mice (aPRGs). Comparing constitutive PACAP knockout mice to a variety of temporally and regionally specific PACAP knockouts, we show that the prominent hyperlocomotor phenotype is a consequence of early loss of PACAP expression, is associated with Fos overexpression in hippocampus and basal ganglia, and that a thermoregulatory effect previously shown to be mediated by PACAP‐expressing neurons of medial preoptic hypothalamus is independent of PACAP expression in those neurons in adult mice. In contrast, PACAP dependence of weight loss/hypophagia triggered by restraint stress, seen in constitutive PACAP knockout mice, is phenocopied in mice in which PACAP is deleted after neuronal differentiation. Our results imply that PACAP has a prominent role as a trophic factor early in development determining global central nervous system characteristics, and in addition a second, discrete set of functions as a neurotransmitter in the fully developed nervous system that support physiological and psychological responses to stress.

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A Low Cost Antibody Signal Enhancer Improves Immunolabeling in Cell Culture, Primate Brain and Human Cancer Biopsy

Cited by 13 publications

References 62 publications

The SLC9C2 Gene Product (Na+/H+ Exchanger Isoform 11; NHE11) Is a Testis-Specific Protein Localized to the Head of Mature Mammalian Sperm

The SLC9C2 Gene Product (Na+/H+ Exchanger Isoform 11; NHE11) Is a Testis-Specific Protein Localized to the Head of Mature Mammalian Sperm

Atrophic astrocytes in aged marmosets present tau hyperphosphorylation, RNA oxidation, and DNA fragmentation

Constitutive and conditional deletion reveals distinct phenotypes driven by developmental versus neurotransmitter actions of the neuropeptide PACAP

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