Liver tissue was fixed in 4% buffered formalin, embedded in paraffin, and sections were mounted onto slides. Sections were dewaxed and then stained with picrosirius red to visualize total collagen, as described[28 (link)]. For immunohistochemistry, tissues were subject to antigen unmasking by heating in citrate buffer (Vector Labs, Burlingame CA), then probed overnight at 4 °C with antibodies directed against type I collagen (ab21286, Abcam, Cambridge, MA) at 1:250 dilution, or α-smooth muscle actin (SMA, clone 1A4, Sigma Chemical, St. Louis, MO) at 1:400 dilution. Positive staining was detected using the DAB Envision System (Dako, Carpenteria, CA). Images were captured and analyzed using ImageJ software as described previously[15 (link)].
Protocol detail
Quantifying Liver Fibrosis by Histology
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Actin
Antibodies
Antigen
Buffer
Citrate
Clone
Collagen
Dilution
Embedded paraffin
Formalin
Immunohistochemistry
Liver
Smooth muscle
Tissues
Type i collagen
Vector
Corresponding Organization : VA Nebraska Western Iowa Health Care System
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Variable analysis
independent variables
- Antigen unmasking by heating in citrate buffer
- Probing with antibodies against type I collagen and α-smooth muscle actin (SMA)
- Total collagen visualization using picrosirius red staining
- Positive staining detection using the DAB Envision System
- Liver tissue fixed in 4% buffered formalin
- Tissue embedded in paraffin
- Sections mounted onto slides
- Sections dewaxed
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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