Browsing by Subject "Histoblot"

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    Developmental regulation of GABAB receptors and downstream molecules in the mouse brain
    (2025) Ana Fajardo Serrano; Rocío Alfaro Ruiz; María Llanos Martínez Poyato; Ana Esther Moreno-Martínez; Sebastián García Madrona; Alberto Roldán Sastre; Pablo Alonso-Gómez; Miriam Fernández; Ricardo Puertas-Avendaño; Ryuichi Shigemoto; Kirill A. Martemyanov; Rafael Luján; Carolina Aguado; Biología Celular e Histología
    Metabotropic GABA (GABAB) receptors have modulatory functions on neuronal excitability and neurotransmitter release. To fulfil these functions, GABAB receptors form macromolecular signaling complexes with G proteins, effectors, and other associated proteins. Here we investigated the postnatal development of GABAB receptors (GABAB1 and GABAB2 subunits) in mouse brain, focusing on potential similarities in the spatial and temporal expression pattern of their associated proteins CaV2.1, Gαo, Gβ5, and RGS7, using histoblots, immunofluorescence, and immunoelectron microscopic techniques. At all ages analyzed, histoblot showed that the six proteins were widely expressed in the brain, with mostly an overlapping pattern throughout postnatal development. In the hippocampus, immunoelectron microscopy and quantitative analysis of immunoparticles for GABAB1, GABAB2, Gαo, Gβ5, and RGS7 revealed their progressive enrichment around excitatory synapses on dendritic spines of CA1 pyramidal cells toward P15. At presynaptic sites, GABAB receptors colocalize with CaV2.1, Gαo, Gβ5, and RGS7 in the active zone and extrasynaptic membranes of axon terminals, establishing synapses on dendritic spines of CA1 pyramidal cells. In the cerebellum, double immunofluorescence at P7 and P10 revealed the colocalization of GABAB1 and CaV2.1 in the whole dendritic tree of developing Purkinje cells. Immunoelectron microscopy at P15 showed that GABAB1, GABAB2, CaV2.1, Gαo, Gβ5, and RGS7 are distributed along the dendritic surface of Purkinje cells, enriched close to excitatory synapses in spines. Altogether, these data suggest that macromolecular complexes composed of GABAB1 /GABAB2/CaV2.1/ Gαo/Gβ5/RGS7 are pre-assembled during key stages of postnatal development in hippocampal and cerebellar neurons.
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    Histoblot: A sensitive method to quantify the expression of proteins in normal and pathological conditions
    (Universidad de Murcia. Departamento de Biología Celular e Histología, 2023) Aguado, Carolina; Martín-Belmonte, Alejandro; Alfaro-Ruiz, Rocío; Martínez Moreno, Ana Esther; Luján, Rafael
    The histoblot (in situ immunoblotting) technique is a simple, reproducible, and sensitive method for protein detection that allows both protein quantitation and analysis of tissue distribution. This easy and fast method allows the direct transfer of native proteins from unfixed frozen tissue sections by mechanical pressure to an immobilizing matrix. Proteins are directly blotted onto nitrocellulose membranes that are then immunolabelled similar to a western blot, but the result is an immunohistochemical imprint of the section retaining all proteins. The histoblot combines advantages of western blot and immunohistochemical methods and yields optimal accessibility of proteins blotted on membranes whilst also preserving anatomical resolution. In addition, it avoids chemical modifications, crosslinking, or semi-denaturation of proteins, which can alter the access of antibody to epitopes, as introduced by conventional immunohistochemistry. Therefore, the histoblot often enables the use of antibodies that do not recognise the target protein in fixed tissue samples. This method has become a trusted alternative to reveal and compare the regional distribution and expression profile of different proteins in the brain in physiological and pathological conditions. In addition, the technique exhibits a high subregional resolution, although is not suitable to unravel protein distribution at the cellular and subcellular levels. In this review, we introduce the histoblot procedure used in our laboratory on brain sections for the identification of quantitative changes of neurotransmitter receptors, ion channels and other signalling molecules in the brain. We also discuss the potentialities, limitations, and fundamental principles of this technique.