Emccd imagem digital camera

Free-floating PN10, PN35 and Adult mouse brain sections from male and female animals (ChAT-Cre/Rosa26^tdTomato/GnRH-GFP) were removed from cryoprotectant (−20°C), rinsed in PBS (1 h), and incubated in blocking solution (2 h, as above). The GnRH-GFP signal was amplified: chicken anti-GFP primary (1:1500 overnight, 4°C, RRID:AB_300798), PBS washes (6x10min), donkey anti-chicken Alexa Fluor 488 (2 h; 1:1000 Jackson Immunoresearch). Sections were mounted on subbed slides, coverslipped with Vectashield Antifade Mounting Media (Vector) and imaged using a spinning disk confocal system CSU10 (Yokogawa), an Eclipse TE200 microscope (Nikon), an EMCCD ImageM digital camera (Hamamatsu), and iVision software (BioVision). Images were processed for viewing using ImageJ (W Rasband, NIH, Bethesda, MD, United States) and Photoshop (Adobe Systems Inc., Salinas, CA) software. Control sections in which one of the primaries was replaced with bovine serum albumin (BSA) showed no detectable signal at that wavelength.

PN10 and adult brain sections from ChAT-Cre/Rosa-Tomato/GnRH-GFP mice were used. Sections were incubated in rabbit anti-p75NGFR (1:5250, 48 h, 4°C, RRID: AB_90760), washed in PBS, incubated in donkey anti-rabbit Alexa Fluor 647-conjugated (2 h; 1:1000; Jackson Immunoresearch), quickly fixed (10 min, 4% formalin), washed (6x5 min PBS) and subsequently stained for GFP (1:1500 overnight, 4°C, RRID:AB_300798) as described above. Sections were imaged with a spinning disk confocal system CSU10 (Yokogawa), an Eclipse TE200 microscope (Nikon), an EMCCD ImageM digital camera (Hamamatsu), and iVision software (BioVision). Images were processed for viewing using ImageJ (W Rasband, NIH, Bethesda, MD, United States) and Photoshop (Adobe Systems Inc., Salinas, CA) software.