Primary neurons were fixed and stained as described previously (Tomita et al., 2003 (link)). After 2–5 months post-injection of AAV, adult mice were deeply anesthetized with pentobarbitul and perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4. After post-fixation, 40 μm sections were prepared using a vibratome (Leica). Sections were incubated with 1 mg/ml pepsin (Dako) in 0.2 N HCl at 37 °C to enhance signal from synaptic proteins (Fukaya and Watanabe, 2000 (link)), followed by staining with appropriate antibodies and imaged using confocal microscopy (Zeiss LSM 710). We acquired data by using Plan-APOCHROMAT 63x/1.40 oil DIC objective lens or Plan-APOCHROMAT 20x/0.8 lens (Zeiss). For quantification, sets of cells and sections were prepared and stained simultaneously. Compared images were acquired at the same time using identical acquisition settings. The numbers of puncta was analyzed using ImageJ (Schneider et al., 2012 (link)) in a blinded manner.
Plan apochromat 20x 0.8 lens
Manufactured by Zeiss
The Plan-APOCHROMAT 20x/0.8 lens is an optical device designed for microscopy applications. It features a high numerical aperture of 0.8 and a flat image field, providing high-resolution imaging capabilities. The lens is optimized for multiple wavelengths, ensuring excellent color correction and image quality.
Automatically generated - may contain errors
2 protocols using plan apochromat 20x 0.8 lens
1
Quantification of Synaptic Protein Puncta
2
Multimodal Visualization of RNA Transcripts
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The RNAscope sample slides were visualized on a LSM 700 laser scanning confocal microscope using a Plan-Apochromat 20x/0.8 lens (air) and 10x objective lens with four different channels (Zeiss Thornwood, NY). Three fluorescent channels were used simultaneously; 1) the green channel 488/520 was assigned for TSA plus ® fluorescein fluorophore (HvGAPDH probe), 2) the blue channel 360/460 was used for DAPI nuclear stain, and 3) the orange channel 550/570 was assigned for TSA plus ® Cyanine3 (Rpg1 probe). T-PMT was assigned for differential interface contrast (DIC) imaging. For multilayer visualization Z-stack images were taken and computation from Z stack was performed in Imaris 9.0.1 software (Bitplane, South Windsor, CT) as described previously [17] (link). During image capture the negative control was assessed first to set the strength of LASER and exposer time. The process from sample collection to image analysis covered a three-day time frame without compromising the integrity of experimental samples (Fig. 3).
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