Tsa plus fluorescein

Wound specimens were fixed in 4% formaldehyde buffered with PBS and then embedded with paraffin. Wound sections were analyzed by triple-color immunofluorescence microscopy as described previously^{4 (link),34 (link)}. Briefly, deparaffinized sections were incubated with PBS containing 1% normal donkey serum and 1% BSA to reduce nonspecific reactions. Then, the sections were further incubated with the combination of anti-mF4/80, -mCD80, and -mCD206 or anti-hCD68, -hiNOS, and -hCD163 Abs at 4 °C overnight. All Abs were used at a concentration of 1 μg/ml. After incubation with HRP-conjugated secondary Abs at room temperature for 30 min and TSA Plus kits (NELL741001KT, TSA Plus Fluorescein, AKOYA Biosciences, Marlborough, MA; or NEL8111001KT, Opal 7-color Manual IHC Kit, PerkinElmer, Waltham, MA) according to the manufacturer’s instruction, the sections were observed by fluorescence microscopy. As negative control, the sections were incubated with non-immunized rabbit IgG or mouse IgG.

RNAscope was performed as described previously following the manufacturer’s protocols (Thomas et al, 2021 (link)). For cells on hydrogels, CPCs were fixed with 10% NBF (HT5011; MilliporeSigma) followed by 3 × 10-min washes with DEPC-treated PBS at 4°C. Cultures were then dehydrated and rehydrated through a series of 10-min ethanol washes at 4°C. Afterward, samples were treated with RNAscope Hydrogen Peroxide Reagent (322330; ACD) for 10 min, rinsed in DEPC-water, and treated with RNAscope Protease III (1:15 in DEPC-PBS) for 10 min followed by DEPC-PBS washes and hybridization (322330; ACD). RNAscope was performed using Multiplex Fluorescent Reagent Kit v2 (323100; ACD). Probes were hybridized to cell culture samples for 2 h at 40°C. Both HCN4 (569141-C1, 1:1; ACD) and NCX1 (1005901-C3, 1:50; ACD) probes were designed by ACD against available NCBI chicken mRNA sequences. All RNAscope assays were performed with a positive control gene UBC (453961-C2, 1:50; ACD). Fluorescent dyes: TSA Plus Fluorescein, (NEL74001KT; Akoya Biosciences), TSA Plus Cyanine 3, (NEL744001KT; Akoya Biosciences), and TSA plus Cyanine 5, (NEL745001KT; Akoya Biosciences) were used at a 1:1,000 dilution in TSA buffer following the ACD RNAscope recommendation.