Phosphate buffered saline (pbs)

Four‐micrometre‐thick paraffin sections were deparaffinized in xylene and sequentially rehydrated using a graded series of ethanol. 100°C EDTA (pH = 9) for 20 min. After rinsing in 1× PBS (Leica) for 15 min (three times, 5 min each time), the sections were blocked with 3% hydrogen peroxide for 10 min. Rinse again with 1× PBS for 15 min. The sections were incubated with cleaved PPARα, FoxO1, eNOS, klotho antibody (1:100) overnight at 4°C. Then, the sections were rinsed with PBS for 15 min and incubated with the appropriate secondary antibodies (Leica) for 15 min at RT. After rinsing with 1× PBS for 15 min, immunoreactivity was detected with 3,3′‐diaminobenzidine (DAB) substrate (Leica) for 10 min and the samples were washed with 1× PBS for 15 min. Haematoxylin dye solution was added to the stain at room temperature for 3 min, and the slides were mounted with neutral gum. All the above steps were performed on Leica Bond RX (Leica). The sections were then viewed by using microscopy (magnification 400×; Leica).

An experimental metastasis experiment was carried out as previously described in [28 (link)]. Briefly, human breast cancer cell lines were engineered by retroviral transduction to stably express GFP. Six to eight-week female CB-17 SCID mice (ARC, Perth, WA, Australia) were injected IV into the tail vein with 5 × 10⁵ cells suspended in 200 µL PBS (Thermo Fisher Scientific, Waltham, MA, USA). Ten weeks after cell injection mice were sacrificed, lungs excised, perfused with PBS and bright field and fluorescent images were recorded by stereo microscope Leica MZ16FA (Wetzlar, Germany).

For IHC, human samples were fixed in 10% buffered formalin and paraffin-embedded. 4 mm-tissue sections were deparaffinized and rehydrated. Novocastra Epitope Retrieval Solution (pH6 or pH9) was used to unmask antigens in a thermostatic bath at 98 °C for 30 min. Subsequently, the sections were brought to room temperature and washed in PBS. After neutralizing the endogenous peroxidases with 3% H₂O₂ and Fcblocking by 0.4% casein in PBS (Novocastra), the sections were incubated with primary antibodies for 90 min at room temperature. The immunostaining was revealed by a polymer detection method (Novolink Polymer Detection Systems Novocastra Leica Biosystems Newcastle Ltd Product No: RE7280-K) and 3,3’-diaminobenzidine (DAB) substrate-chromogen. Slides were analyzed under a Zeiss Axioscope A1 microscope and microphotographs were collected using a Zeiss Axiocam 503 Color digital camera with the Zen 2.0 Software (Zeiss). Slide digitalization was performed using an Aperio CS2 digital scanner (Leica Biosystems) with the ImageScope software (Aperio ImageScope version 12.3.2.8013, Leica Biosystems). Quantitative analyses of IHC stainings were performed by calculating the average percentage of positive signals in five nonoverlapping fields at medium-power magnification (X200) using Nuclear Hub or Positive Pixel Count v9 ImageScope software.

For IHC, human samples were fixed in 10% buffered formalin and paraffin-embedded. 4 mm-tissue sections were deparaffinized and rehydrated. Novocastra Epitope Retrieval Solution (pH6 or pH9) was used to unmask antigens in a thermostatic bath at 98 °C for 30 min. Subsequently, the sections were brought to room temperature and washed in PBS. After neutralizing the endogenous peroxidases with 3% H₂O₂ and Fcblocking by 0.4% casein in PBS (Novocastra), the sections were incubated with primary antibodies for 90 min at room temperature. The immunostaining was revealed by a polymer detection method (Novolink Polymer Detection Systems Novocastra Leica Biosystems Newcastle Ltd Product No: RE7280-K) and 3,3’-diaminobenzidine (DAB) substrate-chromogen. Slides were analyzed under a Zeiss Axioscope A1 microscope and microphotographs were collected using a Zeiss Axiocam 503 Color digital camera with the Zen 2.0 Software (Zeiss). Slide digitalization was performed using an Aperio CS2 digital scanner (Leica Biosystems) with the ImageScope software (Aperio ImageScope version 12.3.2.8013, Leica Biosystems). Quantitative analyses of IHC stainings were performed by calculating the average percentage of positive signals in five nonoverlapping fields at medium-power magnification (X200) using Nuclear Hub or Positive Pixel Count v9 ImageScope software.

For IHC, human samples were fixed in 10% buffered formalin and paraffin-embedded. 4 mm-tissue sections were deparaffinized and rehydrated. Novocastra Epitope Retrieval Solution (pH6 or pH9) was used to unmask antigens in a thermostatic bath at 98 °C for 30 min. Subsequently, the sections were brought to room temperature and washed in PBS. After neutralizing the endogenous peroxidases with 3% H₂O₂ and Fcblocking by 0.4% casein in PBS (Novocastra), the sections were incubated with primary antibodies for 90 min at room temperature. The immunostaining was revealed by a polymer detection method (Novolink Polymer Detection Systems Novocastra Leica Biosystems Newcastle Ltd Product No: RE7280-K) and 3,3’-diaminobenzidine (DAB) substrate-chromogen. Slides were analyzed under a Zeiss Axioscope A1 microscope and microphotographs were collected using a Zeiss Axiocam 503 Color digital camera with the Zen 2.0 Software (Zeiss). Slide digitalization was performed using an Aperio CS2 digital scanner (Leica Biosystems) with the ImageScope software (Aperio ImageScope version 12.3.2.8013, Leica Biosystems). Quantitative analyses of IHC stainings were performed by calculating the average percentage of positive signals in five nonoverlapping fields at medium-power magnification (X200) using Nuclear Hub or Positive Pixel Count v9 ImageScope software.