Tissue tec oct compound

Samples of the optic tectum and spinal cord tissue containing GFP were collected. Embryos of a sufficient size were fixed in 4% formaldehyde solution for 24 hours. Following the fixation, the tissue was immersed in an 18% sucrose solution, embedded in Tissue‐Tec OCT compound (Sakura Finetek, USA), frozen in liquid nitrogen and stored at −80°C until use. Samples were sectioned on a cryotome (Leica 1850, Germany) and the 20 μm thick sections were mounted on poly‐L‐lysine coated slides.

Mice were sacrificed by cervical dislocation, the sciatic nerve was dissected, embedded in Tissue-Tec O.C.T. compound (Sakura Finetek) and frozen in 2-methylbutane at −30°C. Then, 10-μm thick sections were cut using a CM1900 microtome (Leica Microsystems). Frozen sections were shortly thawed and, depending on primary antibody, they were fixed using −20°C cold acetone for 10 min, with 4% paraformaldehyde for 10 min or left unfixed. Following repetitive washing using phosphate-buffered saline (PBS), sections were blocked in PBS containing 5% BSA. Primary antibodies were applied overnight at 4°C, followed by washing in PBS and application of secondary fluorochrome-conjugated antibodies. For nuclear counterstaining DAPI (1 μg/ml; AppliChem) was used. After washing, sections were finally coverslipped with Mowiol/DABCO.
For stainings of cultivated nerve segments, segments were fixed in 4% PFA for 1 h, followed by repetitive washing. Teased fiber preparations were done using fine forceps. Fibers were placed on collagen-coated coverslips, and after permeabilization using 0.3% Triton X-100, staining was performed as stated above.

Human prostate were embedded in Tissue-Tec OCT compound (SakuraFinetek Japan, Tokyo, Japan) and snap frozen. Then tissue was sectioned in 10 μM thick slices and thaw, mounted onto glass slides using a cryostat (Leica CM 1850, Wetzlar, Germany), air-dried, and fixed for 10 min in ice-cold acetone. Slides were washed in PBS and then incubated for 2 h in a mixture of PBS supplemented with 0.2% [v/v] Triton X-100 and 0.1% [w/v] bovine serum albumin, followed by incubation overnight with the primary antibody (rabbit polyclonal to PDE5A, 1:100) and antibody mixture of the PDE5A antibody (1:100) and goat polyclonal to nNOS (1:50, Abcam, ab1376 Cambridge, UK). The secondary antibodies employed to visualize the localization of the two primary antibodies (Jackson ImmunoResearch Inc. West Grove, PA, USA) were Cy3-conjugated goat anti-rabbit IgG (1:1000) and Cy2-conjugated donkey anti-goat IgG (1:400). DAPI was used for staining the nucleus. Negative controls were performed for all samples by omitting the primary antibodies. Human lung tissue was used as a positive control for PDE5A staining. Visualization was done with a laser microscope (Olympus, Tokyo, Japan). Colocalization analysis was performed using NIS-Elements Viewer 3.20 (Nikon, Japan).
All experimental protocol were approved by the research committee of Wuhan University.

Spleens from Egr2-GFP mice and
Foxp3-GFP mice were
rapidly frozen in Tissue-Tec OCT compound (Sakura Finetek, Japan) with liquid
nitrogen, and then were cut into 5-μm sections with a cryostat
microtom. Sections were fixed for 10 min in 4% paraformaldehyde
(Wako) and were preincubated in PBS with 2% BSA and 0.1% saponin, and then
incubated with the following primary antibodies for 30 min: PE
anti-mouse CD4 (5:160),
Brilliant Violet 421 anti-mouse B220 (2:160), FITC anti-GFP (3:160). Images were acquired
with a fluorescence microscope (Olympus BX53). The frequencies of Egr2-GFP⁺CD4⁺ T cells and
Foxp3-GFP⁺CD4⁺ T cells were evaluated by counting the
numbers per field; each field was 0.01 mm².

Histological analysis was conducted at the first, second and third MR measurement point. The aortae were excised and perfused with Tissue Tec O.C.T. Compound (Sakura Finetek Europe B.V., Alphen aan den Rijn, The Netherlands) and stored at -80°C. Serial, transversally cut 8 μm sections of the thoracic aorta were collected at -21°C with a cryotom (Leica CM 1850, Leica Biosystems, Nussloch, Germany). The histological section planes were stained with Hematoxylin and Eosin (HE), Elastica van Gieson and CD68 staining (Merck KGaA, Darmstadt, Germany). For visualization of the elastin laminae and fragmentations the sections were stained with an Elastica van Gieson staining kit. The CD68 coloring (MCA 1957. AbD Serotec, Oxford, UK) is an immunohistochemical staining for macrophages and was performed in an ApoE^-/- mouse after twelve weeks Western Diet.

Tissues were soaked in 30% (wt/vol) sucrose followed by freezing in Tissue-Tec OCT compound (Sakura Finetek, Tokyo, Japan). For histological examination, tissue sections were cut from the frozen block of 5-μm-thick sections, dried and subjected to analysis.