Prostate sections (4–5 μm thick) were de-paraffinized, hydrated, washed with PBS, and incubated in citrate retrieval buffer solution (pH 6.0) for 20 minutes at 100°C followed by treatment with 0.3% hydrogen peroxide in 100% methanol for 20 minutes at room temperature. Immunohistochemistry was performed essentially as described by us previously [38 (link), 39 (link), 41 (link)]. Stained sections were examined under Nikon A1 confocal microscope or Leica microscope equipped with DFC 450C digital camera. Quantitation of immunofluorescence for ACLY, ACC1, and FASN protein expression intensity was performed using ImageJ software. For human prostate tumor tissue array, the H-score was analyzed by positive pixel count V9 algorithm using Aperio ImageScope software.
Protocol detail
Immunohistochemical Analysis of Lipogenic Enzymes
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Acc1
Buffer
Camera
Citrate
Confocal microscope
Fasn
Human
Hydrogen peroxide
Immunofluorescence
Immunohistochemistry
Methanol
Microscope
Prostate
Prostate tumor
Protein
Tissue
Corresponding Organization : UPMC Hillman Cancer Center
Other organizations : University of Pittsburgh
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Variable analysis
independent variables
- Incubation in citrate retrieval buffer solution (pH 6.0) for 20 minutes at 100°C
- Treatment with 0.3% hydrogen peroxide in 100% methanol for 20 minutes at room temperature
- Protein expression intensity of ACLY, ACC1, and FASN
- H-score for human prostate tumor tissue array
- Prostate sections (4–5 μm thick)
- De-paraffinization, hydration, and washing with PBS
- Immunohistochemistry performed as described in previous publications [38, 39, 41]
- Examination of stained sections using Nikon A1 confocal microscope or Leica microscope equipped with DFC 450C digital camera
- Quantitation of immunofluorescence using ImageJ software
- Analysis of H-score using Aperio ImageScope software
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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