P10144

At two sites, 4 mm punches (biopsy puncher, Miltex 33-34) were taken from the whole insert and mounted on cytoslides (Shandon, ThermoFisher 5991057) on ProLong Gold antifade mounting medium (ThermoFisher, P10144). Images were acquired on a Leica SP8 confocal microscope with a 63x oil objective at the BICU imaging unit of Umeå University. Representative sites were imaged as z-stacks of 40 slices with 0.5 μm step size and converted by maximum intensity projection using Fiji (Schindelin et al., 2012 (link)).

The two obtained kinds of ANXs (CD NG, CD + scCO₂ NG) were embedded in OCT embedding glue (SAKURA, Japan), and then frozen sections with a thickness of 6 μm were made using a freezing microtome (CM1900, Leica, USA) on the cross-section of the nerve. At the same time, natural porcine sciatic nerve (Natural SN) was selected as a control. After putting in PBS and degumming, hematoxylin and eosin (H&E) staining was performed according to the kit instructions (G1120, Solarbio, China). The tissue on the section was blocked in 10% goat serum (SL038, Solarbio, China) for 30 min. Laminin antibody (1:1000, L8271, Sigma–Aldrich, USA) from mice was used as the primary antibody to cover the nerve tissue and incubated overnight in the dark at 4 °C. The excess primary antibody was washed off with PBS and then incubated for 2 h with goat anti-mouse IgG H&L secondary antibody (Alexa Fluor 594, 1:200, ab150116, Abcam, USA) in the dark at 37 °C. The excess secondary antibody was washed off with PBS, and sections were incubated with DAPI (1:100, 62248, Thermo Fisher Scientific, USA) staining solution for 5 min in the dark, washed with PBS and mounted with aqueous mount (P10144, Thermo Fisher Scientific, USA). Finally, a microscope equipped with a DP71 camera (BX51, Olympus, Japan) was used to image the stained sections obtained in the above experiment.