6602 Nucleic Acids Research, 2011, Vol. 39, No. 15 described in (40). Brieﬂy, tissue sections were collected on Superfrost/plus slides (Fisher). After washing in two changes of excess PBS, sections were acetylated with 0.1 M triethanolamine/0.25% acetic anhydride for 10 min and then incubated in humidiﬁed bioassay trays for prehybridization at 50 C (20–25 C below the Tm of the probe) for 4 h in hybridization buffer [5 SSC/l Denhardt’s solution/5 mM EDTA/0.1% Tween/0.1% DHAPS/50% deionized formamide/0.1 mg/ml Heparin and 0.3 mg/ml yeast tRNA] (40,41). This was followed by an over-night hybridization step using a DIG-labeled LNA oligonucleotide probe complementary to the target piRNA. Sections were rinsed at a temperature below 50 C and washed twice in 2 SSC and 3 times in 0.2 SSC. The non-speciﬁc binding could be reduced by more stringent washing conditions or by an intense RNase A digestion (10 mg/ml). An in situ signal was detected by incubation with alkaline phosphatase (AP)-conjugated anti-DIG antibody, using NBT/BCIP as substrate. Figure 4A and Supplementary Figures S9–S14 show in situ hybridizations with two different ‘anti-pilRNA’ LNA probes (piRNAa and piRNAb) and four probes corres- ponding to rhesus macaque PIWI transcripts (PIWIL1-4) in adult rhesus macaque tissues. The negative controls shown were subjected to the same hybridization and staining procedure, as the test samples and included alkaline phosphatase (AP)-conjugated anti-DIG antibody, but omitted speciﬁc LNA probes. A negative control shown in Supplementary Figure S12B included scrambled LNA probe in addition to alkaline phosphatase (AP)-conjugated anti-DIG antibody. Western blot For protein extracts used in the western blots, mouse tissues were lysed in RIPA buffer, using Polytron tissue homogenizer. Rabbit anti-Miwi antibody raised against the peptide VIRVPAPCQYAHKLAFLVGQ (amino Figure 4. Localization of pilRNA and Piwi-family mRNA expression in macaque tissues. (A) In situ hybridizations with two different ‘anti-pilRNA’ LNA probes (piRNAa and piRNAb) and probes against four rhesus macaque PIWI transcripts (PIWIL1-4) in adult rhesus macaque tissues. Negative control shows tissue staining after identical hybridization and staining procedure, omitting speciﬁc probes. (B) Rhesus macaque epididymis tissue before and after LCM of principal/basal cells (pilRNA stained) and peritubular tissue (pilRNA free). (C) Small RNA sequencing coverage of pilRNA clusters (red) and background expression clusters (gray) (‘Materials and Methods’ section) in rhesus macaque LCM-puriﬁed principal/basal cells (pilRNA stained) and peritubular tissue (pilRNA free).