Preheated antigen unmasking solution

The alpha-gal epitope was labelled using biotinylated GSL-1 – isolectin B₄ (GSL-1 Lectin; Vector laboratories). Three samples of cellular split-thickness porcine skin, decellularised porcine dermis and decellularised human dermis from three pigs and three human donors were analysed. Paraffin wax sections (5 μm) were dewaxed in xylene and rehydrated through a graded series of alcohols to water. Antigen unmasking was performed by immersing sections in preheated antigen unmasking solution (Vector laboratories) at 95°C for 25 min. Each section was then covered with streptavidin blocking solution and biotin blocking solution (Vector laboratories), each for 15 min. Non-specific binding was then blocked using CarboFree blocking solution (Vector laboratories) for 30 min. GSL-1 Lectin was diluted to 5 μg/mL, added to each section and incubated for 30 min at room temperature. To control sections, galactose-blocked lectin at the same GSL-1 Lectin concentration (5 μg/mL) was added. This was prepared by diluting GSL-1 Lectin in galactose blocking solution (200 mM, 0.1% sodium azide). GSL-1 Lectin was then visualised using streptavidin horseradish peroxidase and ImmPACT DAB detection method.

Formalin-fixed organ samples were paraffin-embedded and sections were stained by H&E for histology. For immunohistochemistry, tissue sections were deparaffinized in xylene and were rehydrated through a series of alcohols and distilled water. Epitope retrieval was performed in preheated antigen unmasking solution (Vector Laboratories, Burlingame, CA, USA) in a water bath. Three percent peroxidase-blocking reagent was applied for 5 minutes. Sections were stained using polyclonal goat anti-mouse IgG1 (dilution 1:2000, A90-105A; Bethyl Laboratories, Montgomery, TX, USA) and biotinylated anti-goat secondary antibody (Vector Laboratories, pk-6105). The detection system consisted of VECTASTAIN ABC reagent (Vector Laboratories, pk-6105) with DAB chromogen (Dako, Glostrup, Denmark). Sections were washed with phosphate-buffered saline three times among each incubation. Sections were counterstained with hematoxylin (Vector Laboratories, H-3401).