Abc elite reagent

Cryosections of 10 μm were prepared from paraffin-embedded epididymal tissues from control (n = 3) and Dicer1 cKO (n = 3) mice. Following deparaffinization, endogenous peroxidase activity was quenched with 3% H₂O₂ (v/v) in methanol for 10 minutes. Sections were washed for 5 minutes in 95% ethanol and 5 minutes in PBS. Non-specific binding sites were blocked with 10% goat serum in PBS for 1 h. Antibodies against AZGP1 were diluted (1:50) in DAKO (Dako North America, Inc.) and applied overnight at 4^°C. For control sections, PBS replaced the primary antibodies. Sections were subsequently incubated with biotinylated goat anti-rabbit antibody for 60 min, and with ABC elite reagent (Vector Laboratories, Inc. Burlingame, Ca) for 30 min. Immunostaining was revealed using 3-amino-9-ethylcarbazole (AEC). Mayer’s haematoxylin solution was used for counterstaining, and mounted under cover slips using an aqueous mounting medium (Sigma). Slides were observed under a Zeiss Axioskop2 Plus microscope linked to a digital camera from Qimaging. Images were captured using the QCapture Pro (Qimaging Instruments).

Floating sections were pretreated with 0.5% H₂O₂ and 0.2% Triton X-100. Cholinergic neurons were detected with the AB144P goat ChAT antiserum (1:2,000; 24h; Merck) (7 (link), 13 (link)), followed by biotinylated secondary antibodies (donkey anti-goat IgG; 1:500; 1h; Jackson ImmunoResearch), ABC Elite reagent (Vector; 1:1,000; 1h) and Cy3-tyramide (diluted 1:1,000 with 0.05M Tris-HCl buffer, pH 7.6, containing 0.005% H₂O₂; 30 min) (14 (link)). Dual-labeled sections were mounted on glass slides, coverslipped with Mowiol and analyzed with confocal microscopy.

All procedures have been described in detail previously [20 (link)]. Briefly, after an overdose of anesthetics, animals were transcardially perfused with physiological saline followed by 4 % PFA in 0.1 M phosphate buffer, pH 7.4. Brains were dissected from the skulls and postfixed overnight. Before sectioning from a dry ice-cooled copper block on a sliding microtome (Leica), brains were transferred to 30 % sucrose in 0.1 M phosphate buffer, pH 7.4. Brains were cut in the coronal plane in 40 μm thick sections. Sections were stained free floating. Immunohistochemistry followed the peroxidase method with a biotinylated secondary antibody (1:500; Jackson ImmunoResearch Laboratories, West Grove, PA), ABC Elite reagent (Vector Laboratories, Burlingame, CA) and diaminobenzidine (DAB; Sigma) as chromogen. For immunofluorescence, FITC- and RhodX-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) were all used at a concentration of 1:250. Primary antibodies were applied in the following concentrations: anti-NeuN (mouse, 1:100; Chemicon, Temecula, CA), anti-desmin (mouse, 1:100, #6322, Abcam), anti-Glucose Transporter-1 (anti-Glut-1; rabbit, 1:100, #400060, Calbiochem).

Positive control experiments with IF double-labeling used two sequential rounds of tyramide signal amplification (TSA) to maximize both GnRH signals. Sections from two subjects (#4 and 5) were pretreated as above, followed by an additional Sudan Black step (Mihály et al., 2000 (link)) to quench tissue autofluorescence. Then, a mixture of guinea pig GnRH (#1018; 1:30,000) and rat GnRH (#1044; 1:20,000) primary antibodies was applied to the sections for 48 hr at 4°C, followed by peroxidase-conjugated anti-guinea pig IgG (Jackson ImmunoResearch; 1:250; 1 hr) and Cy3-tyramide (Hopman et al., 1998 (link)) (diluted 1:1000 with 0.05 M Tris-HCl buffer/0.005% H₂O₂, pH 7.6, 30 min). Peroxidase was inactivated with 0.5% H₂O₂ and 0.1 M sodium azide in PBS for 30 min. Then, the rat GnRH antibodies were reacted with biotin-conjugated secondary antibodies (Jackson ImmunoResearch; 1:500; 1 hr), ABC Elite reagent (Vector; 1:1000; 1 hr), and FITC-tyramide (Hopman et al., 1998 (link)) (diluted 1:1000 with 0.05 M Tris-HCl buffer/0.005% H₂O₂, pH 7.6, 30 min). The dual-labeled sections were mounted and coverslipped with the aqueous mounting medium Mowiol.

Testis and epididymis were collected from C57BL/6 mice, fixed in 4% PFA and embedded in paraffin. Five-μm paraffin sections were deparaffinized in xylene and antigen was unmasked using citrate buffer (10 mM, pH 6.0) for 10 min at 110°C. Protein binding sites were blocked with 0.5% bovine serum albumin (BSA) (Sigma, Canada) for 1 h. Primary antibodies diluted in DAKO solution (Agilent, United States) were incubated overnight at 4°C (Supplementary Table S2). After washing twice with PBS, the secondary biotinylated antibodies were incubated for 1 h at room temperature (Supplementary Table S2). ABC elite reagent (Vector Laboratories, Inc. Burlingame, Canada) was applied to the slides following staining with 3-amino-9-ethylcarbazole (AEC) (Sigma, United States) and hematoxylin (Fisher Chemical, Canada). After addition of aqueous mounting medium (Sigma, Canada), tissue sections were observed under a Zeiss Axioskop 2 Plus microscope linked to a digital camera from Qimaging. Images were captured using the QCapture Pro (Qimaging Instruments).