Protocol detail

Quantitative IHC Evaluation of Kidney Fibrosis

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The IHC staining was carried out as described^{35 (link)–37 (link)}. Briefly, mouse kidney tissues were paraffin-embedded, sectioned, and deparaffinized. After deparaffinization, rehydration and antigen retrieval, sections were blocked and incubated with rabbit anti-Col1a antibody (Abcam), rabbit anti-fibronectin antibody (Abcam), or rabbit anti-α-Sma antibody (Abcam), followed by incubation with a biotin-labeled goat anti-rabbit secondary antibody and streptavidin-conjugated horseradish peroxidase (HRP). The protein of interest was visualized by 3,3′-diaminobenzidine (DAB) staining^{38 (link)–41 (link)}. Rabbit IgG and minus primary antibody stains were used negative controls. Quantitative evaluation of staining results was performed using Image Pro Plus 6.0 software by examining >10 random high-power fields (i.e., 200x magnification) of each sample. The average positive staining area was calculated as the percentage of the total area.

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Yu X., Xia Y., Zeng L., Zhang X., Chen L., Yan S., Zhang R., Zhao C., Zeng Z., Shu Y., Huang S., Lei J., Yuan C., Zhang L., Feng Y., Liu W., Huang B., Zhang B., Luo W., Wang X., Zhang H., Haydon R.C., Luu H.H., He T.C, & Gan H. (2018). A blockade of PI3Kγ signaling effectively mitigates angiotensin II-induced renal injury and fibrosis in a mouse model. Scientific Reports, 8, 10988.

Publication 2018

rabbit anti-fibronectin antibody rabbit anti-α-Sma antibody

Anti antibody Antibody Antigen Biotin Fibronectin Goat Kidney Mouse Paraffin Protein Rabbit Rehydration Streptavidin conjugated horseradish peroxidase Tissues

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Variable analysis

independent variables

IHC staining protocol
Primary antibodies (rabbit anti-Col1a, rabbit anti-fibronectin, rabbit anti-α-Sma)

dependent variables

Staining intensity/area of Col1a, fibronectin, and α-Sma proteins

control variables

Mouse kidney tissues
Paraffin-embedding, sectioning, and deparaffinization
Rehydration and antigen retrieval
Blocking and incubation with secondary antibody (biotin-labeled goat anti-rabbit)
Incubation with streptavidin-conjugated horseradish peroxidase (HRP)
DAB staining to visualize proteins of interest

positive controls

Rabbit IgG

negative controls

Minus primary antibody stains

Annotations

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