Sp5 confocal imaging system

Cells were treated with BMP-2 for 48 hours in dye-free media supplemented with 10% FBS, centrifuged prior to resuspension in calcium-free modified Tyrode buffer (145 mM NaCl, 5.6 mM KCl, 100 μM EGTA, 1.0 mM MgCl₂, 10 mM glucose, and 5.0 mM HEPES, pH 7.2), and incubated for an additional 15 minutes. Fluorescence was measured at room temperature via Leica SP5 confocal imaging system. Two-dimensional confocal images were taken at 1.5 second time intervals. Fluo-4 AM (Dojindo, Japan) was excited at wavelength 488 nm, and emission was detected at 515 nm. Changes in [Ca²⁺]ⁱ were expressed as R = F/F₀, where R is resting fluorescence (F) divided by normalized fluorescence (F₀).

The surface sarcolemma including t-tubules of isolated myocytes were stained by incubating in di-8-ANEPPS at 5 μM for 5 min, then pelleted by centrifugation at approximately ×100 g and re-suspended in Tyrode’s solution. Cells were visualized on a Zeiss Pascal LSM5 laser scanning confocal microscope using a 1.4 NA 63× objective lens with the pinhole set to 1 Airy unit. Di-8-ANEPPS was excited at 488 nm and light collected at wavelengths greater than 505 nm.
Tissue slices were stained with Alexa-Fluor® 488-conjugated wheat-germ agglutinin (WGA) and Alexa-Fluor® 633-conjugated phalloidin. Microscope slides were allowed to warm to RT before adding 5 μg/ml WGA over the tissue sections. WGA was left for 1 h at RT before washing in PBS. Tissue was then stained with phalloidin at 5 U/ml for 1 h at RT before washing again and leaving to air dry. Slides were mounted with Vectashield® containing DAPI and sealed with nail polish. Tissue slices were visualized on a Leica SP5 confocal imaging system using a 1.4 NA 63× objective lens equipped with 405, 488 and 633 nm lasers with the confocal aperture set to 1 Airy unit. DAPI, Alexa-Fluor® 488 and 633 emissions were detected between 400–480, 500–550 and 650–700 nm, respectively.

Sural nerve specimens were fixed overnight in 2% paraformaldehyde-lysine-periodate and then changed to phosphate buffer for storage. Frozen sections (10 μm) were immunostained following established protocols (Hsieh et al., 2008 (link)). Briefly, nonspecific binding was blocked by 0.1% Triton X-100 and 0.5% nonfat milk in Tris buffer, and nerve sections were incubated at 4°C overnight with primary antibodies suspended in 0.1% Triton X-100 and 0.5% nonfat milk in Tris buffer. After the sections were rinsed with Tris buffer, they were incubated with secondary antibodies at room temperature for 1 h. After a rinse in Tris buffer, 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) was used for nuclear staining, if necessary. Fluorescent samples were viewed and scanned under a Leica SP5 confocal imaging system.
For fibrin quantification, the proportion of fibrin(+) blood vessels was derived according to the number of fibrin(+) blood vessels divided by the total number of blood vessels in that nerve tissue. For CD40 quantification, CD40(+) cells in the endoneurium of all nerve fascicles were counted and divided by the area of endoneurium and were expressed as cells/mm².