Bx53 microscope

Mice were perfused intravascularly with 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1M cacodylate buffer pH 7.4. Sciatic nerves and nerve roots were removed and incubated in the same buffer for 90 min at room temperature then overnight at 4°C, with rotation. Specimens were post-fixed for 1 hr with OsO₄, dehydrated then embedded in araldite. Semi-thin (0.5 μm) and ultrathin (70 nm) section were cut using an ultramicrotome, and stained with toluidine blue or uranyl acetate and lead citrate, respectively. Semi-thin sections were examined using bright-field on an Olympus BX53 microscope, and images captured using Hamamatsu ORCA R2 C10600 digital camera and Metamorph software.
For analysis of the number of myelin profiles in control vs mutant sciatic nerves, semi-thin sections from proximal-, mid- and distal regions of the sciatic nerve were taken, and two non- overlapping images of each section were taken using the 60x objective: these images were termed ‘field of view’ (FOV). The average number of myelin profiles in each FOV was calculated, using ImageJ software. For electron microscopy, stained ultrathin sections were examined using a JEOL 1010 electron microscope fitted with a Hamamatsu digital camera and AMT Advantage image capture software.

Photomicrographs of brightfield specimens were captured using a CCD camera (DP-73, Olympus, Tokyo, JAPAN) equipped with a BX-53 microscope (Olympus) using 4× (numerical aperture [N.A.] 0.13), 10× (N.A. 0.3), 40× (N.A. 0.75), 60× (N.A. 0.9) or 100× (N.A. 1.4; oil-immersion) objectives. Photomicrographs were analyzed using Fiji (a distribution of Image J) (Schindelin et al., 2012 (link)), and Adobe photoshop (Adobe Systems Incorporated, Sam Jose, CA, USA). The brightness of digitized images was adjusted using the adjust-level function of these applications. To obtain a multifocus image, images were captured with 1 μm steps and processed with the ‘extended depth of focus’ Fiji plugin. Fluorescent images were captured using an Orca Spark CMOS camera (Hamamatsu photonics, Hamamatsu, Japan) or a DP-73 camera equipped with a BX-53 microscope. To quantify the molecular expression patterns of GP neuron types, immunofluorescent images were acquired using a confocal microscope (FV1200, Olympus) with 40× (N.A. 0.95) or 100× (N.A. 1.35; silicon oil immersion) objectives.

Mice were perfused intravascularly with 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M cacodylate buffer pH 7.4. Sciatic nerves were removed and incubated in the same buffer for 90 min at room temperature, then overnight at 4 °C. Specimens were post-fixed for 1 hr with OsO₄, dehydrated, then embedded in araldite. Semi-thin (0.5 μm) and ultrathin (70 nm) sections were cut using an ultramicrotome and stained with toluidine blue or uranyl acetate and lead citrate, respectively. Semi-thin sections were examined using bright field on an Olympus BX53 microscope, and images were captured using Hamamatsu ORCA R2 C10600 digital camera and Metamorph software. For analysis of the number of myelin profiles in control vs mutant sciatic nerves, semi-thin sections from proximal-, mid- and distal regions of the sciatic nerve were taken, and 2 non- overlapping images of each section were obtained using the 60 x objective. For electron microscopy, stained ultrathin sections were examined using a JEOL 1010 electron microscope fitted with a Hamamatsu digital camera and AMT Advantage image capture software.