Vectashield antifade mounting media with dapi

Pluripotency immunofluorescence: iPSCs (P16-26) were fixed in 4% formaldehyde, permeabilized in 0.5% Triton^® X-100 (for non-surface markers) and blocked in 3% bovine serum albumin. Cells were incubated with primary antibodies at 4 °C overnight. Cells were incubated with secondary antibodies at room temperature for one hour. Trilineage immunofluorescence: iPSCs (P10-26) were differentiated using either the StemMACS^™ Trilineage Differentiation Kit, human, (Miltenyi Biotec) or to cardiac mesoderm using the PSC Cardiomyocyte Differentiation Kit (Gibco). Cells were processed for immunocytochemistry using the Human Three Germ Layer 3-Color Immunocytochemistry Kit (R&D Systems) and Alexa Fluor 488-conjugated secondary antibodies were applied where indicated. Slides were mounted in VECTASHIELD^® Antifade Mounting Media with DAPI (Vector Laboratories). Cell imaging was performed using a Keyence BZ-X810 fluorescence microscope with BZ-X800 Viewer software.

Dissected larvae were first fixed with a carbodiimide solution (1-ethyl-3 [3dimethylaminopropyl] carbodiimide hydrochloride (6%; Millipore Sigma, Cat.No. E6383, year 2019)), N-hydroxysuccinimide (1 mM; Millipore Sigma, Cat.No. 130672, year 2019), and 5% DMSO in PBS) for 1 hour at 37°C or overnight at 4°C. A control group was conducted omitting this step to verify that the antigen lacks a free N-terminus. The tissue was then fixed with paraformaldehyde (4%, Millipore Sigma, Cat.No. P6148, year 2018) in PBS for 15 minutes at room temperature. After fixation, tissue was washed with PBS then permeabilized with Triton™ X-100 (0.3%; Millipore Sigma, Cat.No. X100, year 2019) in PBS for 30 minutes at room temperature followed by a 1-hour incubation in blocking buffer, which was comprised of IgG-free bovine serum albumin (BSA, 1%; Jackson ImmunoResearch, Code: 001-000-162, year 2020) and 0.1% Triton™ X-100 in PBS. Primary antibodies were diluted in blocking buffer and applied overnight at 4°C and appropriate secondary antibodies were applied for 2 hours at room temperature. Cold PBS (7.4 pH) was used to wash between each incubation. Labeled tissue was mounted onto glass slides with Vectashield antifade mounting media with DAPI (Vector Laboratories, Cat.No. H-1200, year 2020).

Cryosections (8μm thick) were cut from wounds collected on day 3, 5, 7, and 9 post-wounding. Haemotoxylin and Eosin (H&E) staining was performed in wound sections for evaluation of re-epithelialization. The percentage of re-epithelialization was calculated as [(distance traversed by epithelium from wound edges)/(distance between wound edges) ×100].
For immunofluorescence staining, sections were first blocked with PBS containing 3% BSA then incubated with primary antibodies against F4/80 and Ki67 (BD Biosciences, clone T45–2342 and Abcam, ab15580). After incubating with Rhodamine anti-rat (Jackson Immunoresearch) and Alexa Fluor 488 anti-rabbit secondary antibodies (Abcam), sections were mounted with VECTASHIELD® Antifade Mounting Media with DAPI (Vector Laboratories). Negative control sections were incubated without primary antibodies. Five distinct images were obtained with a 20×/0.5 objective from selected areas of the granulation tissue (two on each side of wounds and center of wounds. Digital images were obtained using a Nikon Instruments Eclipse 80i microscope. Staining was evaluated using Fiji ImageJ: the same threshold was used for all images, connected cells were cleaned by watershed adjustment and then DAPI+F4/80+ and DAPI+F4/80+Ki67+ positive cells were counted.