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. 2015 Sep 4;9:348. doi: 10.3389/fncel.2015.00348

Figure 1.

Figure 1

Immunohistochemical characterization of GnRH and hGAP1 antibodies. The validation of immunohistochemical labeling with the newly generated rat GnRH (EH#1044; A,F,I), sheep GnRH (EH#2000; B,L,O) and mouse hGAP1 (EH#1001; D,E,R) antibodies used a reference guinea pig GnRH antiserum (EH#1018; C,G,J,M,P,S) as positive control in light (A–E) and confocal (F–T) microscopic specimens. Light micrographs of nickel-diaminobenzidine-stained sections illustrate the distribution of GnRH-IR cell bodies in the medial preoptic area (mPOA) of the mouse using the new rat (EH#1044; 1:200,000; A) and sheep (EH#2000; 1:200,000; B) GnRH antibodies and the guinea pig (positive control) antiserum (EH#1018; 1:200,000; C). Note that all three antibodies reveal typical distribution patterns of preoptic GnRH neurons. Antibodies #1001 raised in mouse ascites fluid against the hGAP1 can label intensely the scattered GnRH-IR cell bodies (D) and their processes (E) in the human hypothalamus, including its infundibular nucleus (INF). A series of dual-immunofluorescent experiments confirms that the new antibodies recognize the same perikarya (arrowheads) and axons (arrows) as the reference guinea pig GnRH antiserum. Panels (F–H) illustrate structures dual-labeled with the rat (1:5000)/guinea pig (1:5000) antiserum combination around the organum vasculosum of the lamina terminalis (OVLT) and also in the eminentia mediana (EM; I–K) where hypophysiotropic GnRH axons project. Panels (L–Q) reveal the dual-labeling of GnRH-IR structures in the mPOA (L–N) and the EM (O–Q) using the sheep (1:10,000)/guinea pig (1:5000) antiserum combination. Similarly, GnRH neurons of the human can be dual-labeled with the combined use of the mouse hGAP1 (1:5000) and the guinea pig GnRH (1:5000) antibodies (R–T). Scale bars = 100 μm in (A–C), 16 μm in (D,E), 25 μm in (F–T).