Significant intrinsic fluorescence in tissues and in disassociated cells can interfere with fluorescence identification of target cells. The objectives of the present study were (1) to examine an intrinsic fluorescence we observed in both the piglet testis tissue and cells and (2) to test an effective method to block the autofluorescence. We observed that a number of granules within the testis interstitial cells were inherently fluorescent, detectable using epifluorescence microscopy, confocal laser scanning microscopy, and flow cytometry. The emission wavelength of the autofluorescent substance ranged from 425 to 700？nm, a range sufficiently broad that could potentially interfere with fluorescence techniques. When we treated the samples with Sudan Black B for different incubation times, the intrinsic fluorescence was completely masked after treatment for 10–15？min of the testis tissue sections or for 8？min of the testis cells, without compromising specific fluorescence labeling of gonocytes with lectin Dolichos biflorus agglutinin (DBA). We speculate that the lipofuscin or lipofuscin-like pigments within Leydig cell granules were mainly responsible for the observed intrinsic fluorescence in piglet testes. The method described in the present study can facilitate the identification and characterization of piglet gonocytes using fluorescence microscopy. 1. Introduction The mammalian testis is composed of seminiferous tubules, primarily containing germ and Sertoli cells, and interstitial tissues containing Leydig cells. As the earliest identifiable germ cell progenitors, primordial germ cells (PGCs) proliferate and differentiate in the fetal testis gonad into gonocytes [1–3]. After birth, gonocytes proliferate in the testis and develop into spermatogonial stem cells (SSCs) prior to puberty [4, 5]. In the mature testis, SSCs initiate and maintain the continuity of spermatogenesis through self-renewal, proliferation, and differentiation to produce daughter germ cells eventually leading up to spermatozoa [4, 6]. In the neonatal testis, gonocytes are the only germ cells present [7–11], and although they give rise to SSCs and are considered germline stem cells, there is controversy as to whether gonocytes have the capability to initiate spermatogenesis on their own, that is, without first developing into SSCs [12–16]. Compared with PGCs and SSCs, gonocytes are the least investigated germline progenitor cells [17]; therefore, obtaining new knowledge about gonocytes may also shed light on the germline stem cells as a whole. Although gonocytes can be identified in
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