Quantifying Glomerular Basement Membrane Thickness

Sections from the renal cortex area of the right kidneys were diced into 1–2 mm pieces and fixed in 4% PFA for 4 h followed by post-fixation with 1% osmium tetroxide for 30 min. Then, sections were then dehydrated in graded alcohol, transferred to propylene oxide and embedded in Epon 812 resin in blocks. Eighty nanometer sections were collected using a diamond histoknife (Diatome) on an Ultracut E microtome (Leica), and then mounted on a copper mesh and stained with uranyl acetate and lead nitrate. The UTHSCSA Image Tools 3.0 (University of Texas Medical School at San Antonio) was used to determine the thickness of the glomerular basement membrane. Glomerular basement membrane (GBM) thickness was determined by the orthogonal intercept method.19 (link)

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Xu S., He L., Ding K., Zhang L., Xu X., Wang S, & Qian X. (2020). Tanshinone IIA Ameliorates Streptozotocin-Induced Diabetic Nephropathy, Partly by Attenuating PERK Pathway-Induced Fibrosis. Drug Design, Development and Therapy, 14, 5773-5782.

Publication 2020

diamond histoknife Ultracut E microtome

Copper Cortex of kidneys Dehydrated alcohol Diamond Epon 812 Glomerular basement membrane Lead nitrate Microtome Osmium tetroxide Propylene oxide Renal Resin Uranyl acetate

Corresponding Organization :

Other organizations : Wannan Medical College, First Affiliated Hospital of Wannan Medical College, Southwest Medical University

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Variable analysis

independent variables

Dicing of sections from the renal cortex area of the right kidneys into 1–2 mm pieces

dependent variables

Thickness of the glomerular basement membrane (GBM)

control variables

Fixation of the diced sections in 4% PFA for 4 h
Post-fixation with 1% osmium tetroxide for 30 min
Dehydration of sections in graded alcohol
Transfer to propylene oxide
Embedding in Epon 812 resin in blocks
Collection of 80 nanometer sections using a diamond histoknife on an Ultracut E microtome
Mounting of sections on a copper mesh
Staining with uranyl acetate and lead nitrate

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