Retiga q image digital still camera

Cryosections of urethral tissue samples were prepared as described previously[15 (link)]. Immunofluorescence was performed as described previously [16 (link)] using phalloidin and anti-myosin heavy chain antibody (Abcam Inc., Cambridge, MA, USA). To identify intramyocellular lipid (IMCL) within urethral striated muscle, we employed the staining procedure reported previously [13 (link)]. After washing with PBS, the slides were incubated with LipidTOX neutral lipid stain (1:1000, Invitrogen) at room temperature for 30 minutes, followed by staining with phalloidin (Invitrogen). For image analysis, five randomly selected fields per tissue per animal for each treatment group were photographed and recorded using a Retiga Q Image digital still camera and ACT-1 software (Nikon Instruments Inc., Melville, NY, USA).

Tissues were rehydrated with PBS for 5 minutes, and treated with hydrogen peroxide/methanol to quench endogenous peroxidase activity. After rinsing with water, sections were washed twice in PBS for 5 minutes, followed by 30 minutes of incubation with 3% horse serum/PBS/0.3% triton X-100. After excess fluid was drained, sections were incubated overnight at 4°C with primary antibodies including anti-neuronal nitric oxide synthase (nNOS; 1:200; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), anti-tyrosine hydroxylase (TH; 1:200; Millipore Corp, Bedford, MA, USA), anti-choline acetyltransferase (ChAT; 1:200; Millipore, , Bedford, MA, USA) and anti-myosin heavy chain (MHC; 1:500; Abcam, Cambridge, MA, USA) . Secondary antibodies used included Alexa-488- and Alexa-594- conjugated antibodies (1:500; Invitrogen). Nuclei were stained with DAPI. For image analysis, five randomly selected fields per tissue were photographed and recorded using Retiga Q Image digital still camera and ACT-1 software (Nikon Instruments Inc., Melville, NY).