{"id":58,"date":"2018-01-19T12:01:57","date_gmt":"2018-01-19T11:01:57","guid":{"rendered":"http:\/\/hhrc.koki.hu\/?page_id=58"},"modified":"2018-01-22T14:18:46","modified_gmt":"2018-01-22T13:18:46","slug":"methods","status":"publish","type":"page","link":"http:\/\/hhru.koki.hu\/?page_id=58","title":{"rendered":"Methods"},"content":{"rendered":"

Methods<\/b><\/span><\/h2>\n

1. Immunohistochemistry<\/b><\/span><\/h3>\n

\"1\" Both immersion-fixed and perfusion-fixed hypothalami are used routinely in immunohistochemical studies. The figure <\/b>illustrates the results of double-label immunohistochemical experiments which revealed estrogen receptor beta immunoreactivity (black) in human GnRH neurons (brown).<\/span><\/p>\n

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2. Morphometric analysis of immuno-labeled hypothalamic <\/b>neurons<\/b><\/span><\/h3>\n

\"2\" The large number of the immersion-fixed tissue specimens enables the morphometric studies of neurons to establish sex differences and aging-related changes. The example in the figure <\/b>reveals a significant sex difference in the size (mean profile area) of human kisspeptin neurons in the infundibular nucleus of middle-aged male and female humans. The increased cell size in postmenopausal women is a consequence of a neuronal hypertrophy in the absence of estrogens (for details, see Hrabovszky et al., Front Endocrinol (Lausanne): 2:80).<\/span><\/p>\n

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3. Immunofluorescent multiple-labeling and confocal <\/b>microscopy<\/b><\/span><\/h3>\n

\"3\" This picture illustrates the discovery of cocaine- and amphetamine-regulated transcript (CART) in a subset of kisspeptin (KP) and neurokinin B (NKB)-containing neurons of the human mediobasal hypothalamus (for details, see Skrapits et al., PLoS One: 9(8): e103977).<\/span><\/p>\n

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4. Quantitative immunohistochemistry<\/b><\/span><\/h3>\n

\"4\" The immersion-fixed tissue samples are compatible with the quantitative analysis of neuropeptide coexpression patterns. This figure depicts the results of colocalization experiments between substance P (SP), kisspeptin (KP) and neurokinin B (NKB) immunofluorescent signals within neuronal cell bodies of the human infundibular nucleus (for details of this study, see Hrabovszky et al., PLoS One:8(8): e72369).<\/span><\/p>\n

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5. Preembedding<\/b> immunoelectron<\/b> microscopy<\/b><\/span><\/h3>\n

\"5\" Short post mortem-time immersion-fixed hypothalami are compatible with immunoelectron microscopy. These figures reveal kisspeptin-immunoreactive axons (semi-transparent blue) establishing asymmetric synapses with kisspeptin-immunoreactive cell bodies (semi-transparent red, cb), dendrites (d) and dendritic spines (s) in the human infundibular nucleus (Tak\u00e1cs et al., in press).<\/span><\/p>\n

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6. In situ<\/i><\/b> hybridization with <\/b>radioisotopic<\/b> and digoxigenin-labeled <\/b>c<\/b>RNA probes<\/b><\/span><\/h3>\n

\"6\" Short post mortem-<\/i>time tissues are compatible with in situ<\/i> hybridization experiments. The figures illustrate autoradiographic in situ<\/i> hybridization signal to RF-amide related peptide mRNA in the hypothalamic periventricular nucleus (left) and non-isotopic in situ<\/i> hybridization signal to proGnRH mRNA in the human putamen (right) (in preparation).<\/span><\/p>\n

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7. Random D<\/b>i<\/b>I-labeling and 3D-reconstruction of immunolabeled neurons in <\/b>post mortem<\/i><\/b> brains<\/b><\/span><\/h3>\n

\"7\" This figure illustrates the \u2018diolistic\u2019 labeling of an immunohistochemically identified kisspeptin neuron (green) using a Gene Gun loaded with the lipophilic dye DiI adsorbed to tungsten beads. The DiI signal (magenta) reveals the presence of numerous somatic and dendritic spines in double-labeled kisspeptin neurons (Tak\u00e1cs et al., in press).<\/span><\/p>\n

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8. RT-qPCR <\/b>studies of <\/b>microdissected<\/b> hypothalamic nuclei<\/b><\/span><\/h3>\n

\"8\" The figure <\/b>demonstrates the successful use of micropunched hypothalamic nuclei for RT-qPCR amplification of VGLUT1 and VGLUT2 mRNAs in the human mediobasal hypothalamus (S\u00e1rv\u00e1ri et al., in preparation).<\/span><\/p>\n","protected":false},"excerpt":{"rendered":"

Methods 1. Immunohistochemistry Both immersion-fixed and perfusion-fixed hypothalami are used routinely in immunohistochemical studies. The figure illustrates the results of double-label immunohistochemical experiments which revealed estrogen receptor beta immunoreactivity (black) in human GnRH neurons (brown). 2. Morphometric analysis of immuno-labeled hypothalamic neurons The large number of the immersion-fixed tissue specimens enables the morphometric studies of … Continue reading “Methods”<\/span><\/a><\/p>\n","protected":false},"author":1,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"footnotes":""},"class_list":["post-58","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"http:\/\/hhru.koki.hu\/index.php?rest_route=\/wp\/v2\/pages\/58","targetHints":{"allow":["GET"]}}],"collection":[{"href":"http:\/\/hhru.koki.hu\/index.php?rest_route=\/wp\/v2\/pages"}],"about":[{"href":"http:\/\/hhru.koki.hu\/index.php?rest_route=\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"http:\/\/hhru.koki.hu\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"http:\/\/hhru.koki.hu\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=58"}],"version-history":[{"count":9,"href":"http:\/\/hhru.koki.hu\/index.php?rest_route=\/wp\/v2\/pages\/58\/revisions"}],"predecessor-version":[{"id":141,"href":"http:\/\/hhru.koki.hu\/index.php?rest_route=\/wp\/v2\/pages\/58\/revisions\/141"}],"wp:attachment":[{"href":"http:\/\/hhru.koki.hu\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=58"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}