Browsing by Subject "Alkaline phosphatase"

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    Alkaline phosphatase activity of the IVth ventricular choroidal epithelium of rats during embryonic and neonatal development
    (Murcia : F. Hernández, 1988) Takafumi Yoshioka; Osamu Tanaka; Hiroki Otani; Haruo Shinohara; Kenichirou Inomata
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    An ultracytochemical study on the dynamics of alkaline phosphatase-positive granules in rat neutrophils
    (F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 1998) Jiang, X.; Kobayashi, T.; Nahirney, P. C.; García del Saz, E.; Seguchi, H.
    Alkaline phosphatase (ALPase) activity was examined by cerium-based uitracytochemistry in isolated rat neutrophils following stimulation with phorbol myristate acetate (PMA) or N-formylmethionyl-leucyl-phenylalanine (fMLP). In control neutrophils, low levels of ALPase activity were detected in small tubular and spherical compartments distributed throughout the cytoplasm. Neutrophils stimulated for 2.5, 5, 15, and 30 min with 50 nglml PMA or 10-7 M fMLP displayed a time-dependent increase in ALPase activity. At 2.5 min, an increase in activity was first identified in compartments that were aggregated in the central regions of the cell. By 15 min, a dense precipitate was seen in tubular or elongated bead-like structures that extended to and made contact with the plasma membrane. Large enzyme-positive vacuoles were also observed in regions near the plasma membrane. At the longer stimulation times, a fine precipitate was present on the cell surface of the neutrophil in regions where subplasmalemmal ALPase activity was present. The results of this study indicate that an increase in activity and a redistribution of ALPase-positive structures occurs in neutrophils in response to stimulation with PMA and fMLP. It is likely that these compartments are latent pools of ALPase which, upon stimulation, fuse and mobilize the enzyme activity to the cell surface.
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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.