Ultravision lp large volume detection system hrp polymer

The dorsal skins were fixed with 10% formalin immediately after collection, gradually dehydrated in 80%, 90% and 100% ethanol and cleaned with xylene. Next, the tissues were embedded in paraffin, and hematoxylin and eosin (H&E) staining was carried out. Masson’s trichrome staining was performed for the detection of collagen fibers in the skin tissue. The paraffin-embedded tissues were placed in a 5% iron alum solution for 30 min at 56 °C and then stained with Weigert iron hematoxylin and Biebrich scarlet acid fuchsin solution. After the first stain, the slides were placed in a phosphomolybdic-phosphotungstic acid solution and then stained with aniline blue solution. The stained slides were observed using a light microscope (Leica DM750, Wetzlar, Germany) to detect collagen fibers and nuclei. Immunohistochemistry (IHC) was performed to analyze LAMP-1 and collagen-1 using a UltraVision LP large volume detection system HRP polymer (TL-060-HL, Thermo Scientific) according to the manufacturer’s protocol. LAMP-1/CD107a antibody (1:500, NB120-19294, Novus Biologicals, Centennial, CO, USA) and collagen-1 antibody (1:500, NB600-408, Novus Biologicals) were used as primary antibodies and goat antirabbit IgG secondary antibody (HRP (horseradish peroxidase)) (1:1000, HAF008, R&D Systems, Minneapolis, MN, USA) was used as the secondary antibody.

Formalin-fixed paraffin-embedded tissue (FFPE) sections were deparaffinized in an incubator at 65°C for 40–60 minutes followed by incubation in xylene (5 min +10 min, twice) and xylene-EtOH mixture (1∶1) for another 5 min. Sections were rehydrated by incubation in decreasing alcohol concentrations. Microwave treatment at 90W for 20 min in 10% target retrieval solution pH 6 (Dako Österreich GmbH) was followed by endogenous peroxidase blocking through incubation in 3% H₂O₂ for 10 min. To reduce unspecific antibody binding, blocking with serum-free protein block (Dako Österreich GmbH) was performed for 30 min. Sections were incubated with anti-proteolipid protein (PLP) antibody (1 µg/ml) (Chemicon (Millipore)) and isotype control (1 µg/ml) overnight (+4°C). Sections were subsequently incubated with a secondary antibody anti-IgY (host rabbit) (Abcam) for 1 hour and reactivity was visualized with UltraVision LP large-volume detection system HRP polymer (Thermo Fischer Scientific, Freemont, CA, www.labvision.com) and stained with Dako REAL DAB+ Chromogen (Dako Österreich GmbH) for 1 min following the manufacturer's instructions. Stained sections were counterstained with Harriś Hematoxylin solution (Merck) for 20 seconds, blued in hot tap water and mounted with mounting medium (Tissue Tek, Coverslipping Resin, Sakura Finetek USA Inc, Torrance, CA; www.sakuraus.com).