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Role and Gonadotrophic Regulation of X-Linked: MATERIALS AND METHODS(7)

METHODS(7)

To confirm the success of adenoviral infection, follicles injected with the adenoviral XIAP sense cDNA or LacZ were paraffin-sectioned and processed with the same procedure as described in the above section on the TUNEL assay up to the incubation step with the blocking reagents. Sections were then incubated (2 h at RT or overnight at 4°C) with HRP-conjugated anti-Myc antibody (1:50 [v/v]; Invitrogen) or rabbit anti-p-galactosidase (1:100 [v/v]; ICN Pharmaceuticals, Inc., Biochemical Division, Aurora, OH), respectively. Sections of LacZ-infected follicles were washed in PBS, incubated with second antibody (HRP-conjugated goat anti-rabbit IgG, 1:200 [v/v] in PBS, 1 h, RT) and detected by diamino-benzidine (DAB) staining (4 min; Boehringer Mannheim Corp., Indianapolis, IN) and counterstained with Methyl Green (100%, v/v; Vector Laboratories). The Myc-tag in sections of XIAP sense-injected follicles were directly detected with DAB staining after incubation with primary antibody. cialis canadian pharmacy

In addition, adenoviral LacZ-infected follicles (MOI = 20; 3 days) were washed twice with PBS, fixed with glutaraldehyde (0.25% [v/v] in PBS, 10 min, 4°C), rinsed four times with PBS, and stained in 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) buffer (PBS [pH 7.27.5] containing X-gal [1 mg/ml], K3Fe[CN]6 [5 mM], K4Fe[CN]6 [5 mM], MgCl2 [2 mM], and Triton X-100 [0.05%, v/v]; 18 h; 37°C). Stained follicles were washed twice with PBS and then incubated in a series of glycerol concentrations (10%, 20%, 40%, and 60% [v/v]; 4 h for each concentration; RT).

Successful infection of follicular cells with adenoviral LacZ and myctagged XIAP sense cDNA was confirmed by X-gal staining (on intact follicles) (Fig. 3, A and B) and immunohistochemistry (IHC; on follicular sections) using anti-galactosidase (87%) (Fig. 3, C and D) and anti-Myc (82%) (Fig. 3, E and F) antibodies.