Immunostaining of Renal Fibrosis Markers

Paraffin sections were stained with antibodies against fibronectin, collagen I, and α-SMA. After fixation and antigen retrieval, nonspecific binding was blocked with protein block (Dako, Carpinteria, CA). Kidney sections were then incubated with rabbit anti-collagen I antibody (Rockland Immunochemicals, Gilbertsville, PA), rabbit anti-fibronectin antibody (Sigma-Aldrich, St. Louis, MO), or rabbit anti-α-SMA antibody (Abcam, Cambridge, MA) followed by Alexa-488 conjugated donkey anti-rabbit antibody (Invitrogen, Carlsbad, CA). For double immunofluorescence staining, renal tissues were embedded in OCT compound, snap-frozen on dry ice, cut at 5 μm thickness, and mounted. Kidney sections were fixed and stained with rat anti-CD45 (BD Biosciences) and platelet-derived growth factor receptor (PDGFR)-β (Santa Cruz Biotechnology) followed by appropriate secondary antibodies sequentially. Slides were mounted with medium containing DAPI. Fluorescence intensity was visualized using a microscope equipped with a digital camera (Nikon Instruments Inc., Melville, NY). Quantitative evaluation of sections stained with antibodies to α-SMA, collagen I and fibronectin was performed using NIS-Elements Br 3.0 software. The fluorescence positive area was calculated as a percentage of the total area as described10 (link)12 (link)21 (link).

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Liang H., Ma Z., Peng H., He L., Hu Z, & Wang Y. (2016). CXCL16 Deficiency Attenuates Renal Injury and Fibrosis in Salt-Sensitive Hypertension. Scientific Reports, 6, 28715.

Publication 2016

protein block rabbit anti-collagen I antibody rabbit anti-fibronectin antibody rabbit anti-α-SMA antibody Alexa-488 conjugated donkey anti-rabbit antibody rat anti-CD45 PDGFR)-β digital camera

Anti antibody Antibodies Antigen Camera Collagen i Dapi Donkey Dry ice Fibronectin Fluorescence Frozen Immunofluorescence Kidney Microscope Paraffin Platelet derived growth factor receptor Protein Rabbit Tissues

Corresponding Organization :

Other organizations : Baylor College of Medicine, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine

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Variable analysis

independent variables

Antibodies used for staining (fibronectin, collagen I, and α-SMA)

dependent variables

Fluorescence intensity of stained kidney sections
Quantitative evaluation of stained areas (α-SMA, collagen I and fibronectin) as a percentage of total area

control variables

Fixation and antigen retrieval methods
Blocking of nonspecific binding with protein block
Incubation with primary and secondary antibodies
Mounting medium containing DAPI

controls

Positive control: None specified
Negative control: None specified

Annotations

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