Shandon cryotome fse

On the 7th day after ischemic stroke, rats (n = 6) in each group were anaesthetized with 10 % chloral hydrate. Brains were fixed by transcardial perfusion with saline, followed by perfusion and immersion in 4 % paraformaldehyde. Fresh frozen sections of 5 μm thickness were cut on a cryostat microtome (Thermo fisher Scientific Shandon Cryotome FSE). Every 5th coronal section for a total of 6 sections was used for immunofluorescence staining. Prior to all staining procedures, tissue slices were extensively rinsed with 0.02 M PBS and blocked with 5 % normal goat serum in 0.02 M PBS containing 0.3 % Triton X-100 for 1 h. To identify the expressions of VEGF and BDNF in the IBZ, the slices incubated with rabbit primary antibodies to VEGF (1:50) and BDNF (1:200) at 4 °C overnight, followed by secondary antibodies to Alexa Fluor 488 labeled goat anti-rabbit IgG (H + L) (Life Technologies, USA) at 37 °C for 1 h. Nuclei were finally stained with 4′,6-diamidino-2-phenylindole in all images. The slices were visualized and digitally photographed with a confocal laser scanning microscope (Zeiss LSM710, Germany), 5 non-overlapping fields of one slice were randomly observed under a magnification of 10 × 40 in confocal images. To quantitatively analyze the proteins expression, Image-Pro Plus software was applied.

Soleus muscle samples were cut in 10 µm sections from the midbelly of the muscle using a cryostat (Thermo Scientific, Shandon Cryotome FSE) and allowed to air dry for 30 min. We used NADPH diaphorase histochemical analysis described by Hord, et. al [17 (link)] to ascertain the presence and localization of active nNOS. Soleus muscle cross-sections were fixed for 20 min with 2% paraformaldehyde, then washed 2X in PBS. Slides were then incubated at 37 °C for two hours in 50 mM Tris-HCl buffer (pH = 8.00) with 0.2% Triton-X-100, 0.5 mM nitrotetrazolium blue chloride, and 1 mM β-NADPH. Distilled water was used to stop the enzymatic reaction, and slides were then air dried. Soleus sections were then mounted with Vectamount permanent medium (Vectamount, Cat# H-5000, Vector Laboratories, Burlingame, CA, USA).
Images of stained were taken using a Zeiss Axioplot upright microscope and Zeiss Axiocam HRc color camera (Carl Zeiss Microimaging, Thornwood, NY, USA). NIH Image J was used to quantify NADPH-diaphorase-stained samples ImageJ by using the cross-sectional circumference (CSC) to quantify the percentage of the positively stained sarcolemma.

Wild-type mice were anesthetized (2.5% isoflurane in O₂ at 1 L/min flow rate) and intravenously injected with [¹⁸F]AlF-NOTA-DV1-k-(DV3) and [⁶⁸Ga]Ga-DOTA-DV1-k-(DV3) (3.5–4 MBq). After 75 min, mice were sacrificed by decapitation. Organs of interest were quickly excised and snap frozen in 2-methylbutane (−40 °C). Next, 20 μm sections of liver, kidney and spleen were obtained using a cryotome (Shandon cryotome FSE; Thermo Fisher Scientific, Waltham, MA, USA) and these were mounted on adhesive microscope slides (Superfrost Plus; Thermo Fisher Scientific, Thermo Fisher Scientific, Waltham, MA, USA) after which they were exposed to a phosphor storage screen (super-resolution screen; Perkin Elmer, Waltham, MA, USA) overnight. Screens were read in a Cyclone Plus system (Perkin Elmer, Waltham, MA, USA), and images were analyzed using OptiQuant software (Perkin Elmer, Waltham, MA, USA). The remaining excised tissue was further sectioned in 20 μm slices and stored at −20 °C.