Tsa kit

To verify proliferation of the PDL tissue as a result of the orthodontic force, we used rat polygonal primary antibodies for proliferating cell nuclear antigen (PCNA), and immunofluorescence staining was performed using a tyramide signal amplification (TSA) kit (Invitrogen, Carlsbad, CA, USA). The 5-µm-thick sections were obtained from the tissues of both the experimental and the control groups. Each section was placed on a slide, fixed with 4% paraformaldehyde for 20 minutes, and then washed with phosphate-buffered saline for 5 minutes. We used 3% H₂O₂ to block endogenous peroxidase for 1 hour, and the sections were reacted with the primary antibody for 1 day, followed by reaction with biotinylated secondary antibody for 1 hour. Tyramide working solution was used to colorize the reactants. The reactants were visualized and photographed using a confocal laser scanning microscope (LSM) (Carl Zeiss, Göttingen, Germany). Immunological specificity was assessed by substituting the primary antibody from the normal serum.

FFPE normal pancreas tissue sections (5-μm thick) were mounted on charged slides and baked at 60°C for 1 hour, deparaffinized, and dehydrated. Slides were then washed with deionized water for 2 minutes followed by antigen retrieval at 96°C with 10 mmol/L sodium citrate (pH 6.0) for 8 min. Once the slides cooled to room temperature, they were washed three times (2 minutes per wash) with PBS and endogenous peroxides were quenched with 3% hydrogen peroxide for 15 minutes. The TSA kit (Invitrogen) was used according to the manufacturer's instructions. Briefly, slides were incubated with blocking serum provided in the kit and stained with antigen-specific primary antibodies overnight at 4°C: Claudin18 (1:100), AQP1 (1:500), AQP3 (1:200), MUC5AC (1:200), or MUC5B (1:200). The next day, slides were washed three times with PBS and incubated in anti-rabbit secondary antibody for 1 hour. This was followed by an additional three washes with PBS. TSA conjugated to 488 or 555 fluorophores was applied for 10 minutes, followed by incubation with a reaction stop solution for 5 minutes. Finally, slides were incubated with DAPI for 5 minutes and washed three times with PBS prior to being mounted with ProLong Diamond Antifade. High-magnification images were obtained using confocal microscopy (Leica Stellaris 8). Antibodies used are listed in Supplementary Table S5.

Formalin-fixed, paraffin-embedded (FFPE) lung tissue sections (5–10 μm thick) were mounted on charged slides and baked at 60°C for 1 hour, deparaffinized, and dehydrated. Slides were then washed with deionized water for 2 minutes followed by antigen retrieval at 96°C in 10 mmol/L sodium citrate (pH 6.0) for 8 minutes. Once cooled to room temperature, slides were washed three times (2 minutes per wash) with 1x PBS and endogenous peroxides were quenched with 3% hydrogen peroxide for 15 minutes. The TSA kit (Invitrogen) was used per the manufacturer’s guidelines. Briefly, slides were incubated with proprietary blocking serum provided in the kit then stained with antigen-specific primary antibodies overnight at 4°C: phospho-ERK (1:200), PDGFRb (1:100), aSMA (1:1000), F4/80 (1:200), ECAD (1:100). The following day, slides were washed three times with 1x PBS then incubated in anti-rabbit secondary antibody for 1 hour at room temperature. Slides were then washed three more times with 1x PBS. TSA conjugated to 488 or 555 fluorophores was applied for 10 minutes, followed by incubation with a reaction stop reagent for 5 minutes. Finally, slides were incubated with DAPI for 5 minutes followed by three washes with 1x PBS prior to mounting with ProLong Diamond Antifade mounting solution. Antibodies used are listed in Supplemental Table 1.

After 14 days of neuronal differentiation, cells were fixed with 10% neutral formalin for 24 h and immunofluorescence staining was done using the tyramide signal amplification (TSA) Kit (Invitrogen, Carlsbad, CA, USA). Briefly, after blocking endogenous peroxidase, cells were reacted with the NFM antibody (Biolegend) overnight and subsequently with the horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology). Cells were incubated in Alexa Fluor 488 tyramide solution (Thermo Fisher Scientific, Waltham, MA, USA) and then counterstained with propidium iodide (Thermo Fisher Scientific). Cells were photographed using an LSM confocal microscope (Carl Zeiss, Oberkochen, Germany).