Four micrometer thick formalin-fixed paraffin-embedded sections of WHO grade I MG samples from 11 patients were subjected to 3,3-diaminobenzidine (DAB) IHC staining for (pro)renin receptor (PRR) (1:2000; cat# ab40790, Abcam, Cambridge, UK), ACE (1:100; cat# MCA2054, AbD Serotec, Kidlington, UK), ATIIR1 (1:30; cat# ab9391, Abcam and ATIIR2 (1:2000; cat# NBP1-77368, Novus Biologicals, LLC, Littleton, CO, USA). Surgipath Micromount (Leica) was used to mount all the slides. Staining of MG sections with a mouse (ready-to-use; cat# IR750, Dako, Copenhagen, Denmark) and rabbit (ready-to-use; cat# IR600, Dako) primary antibody isotype control combination was performed as an appropriate negative control, as previously described (24 (link)).
Two of the MG samples subjected to DAB IHC staining underwent immunofluorescence (IF) IHC staining using combinations of CD34 (ready-to-use; cat# PA0212, Leica), ERG (ready-to-use; cat# EP111, Cell Marque, Rocklin, CA, USA) (25 (link)), and OCT4 (1:30; cat# MRQ-10, Cell Marque) that highlighted the endothelium and stem cells on the microvessels, respectively (13 (link)). Appropriate positive human control tissues included placenta for PRR, liver for ACE and ATIIR1, and kidney for ATIIR2.
Two of the MG samples subjected to DAB IHC staining underwent immunofluorescence (IF) IHC staining using combinations of CD34 (ready-to-use; cat# PA0212, Leica), ERG (ready-to-use; cat# EP111, Cell Marque, Rocklin, CA, USA) (25 (link)), and OCT4 (1:30; cat# MRQ-10, Cell Marque) that highlighted the endothelium and stem cells on the microvessels, respectively (13 (link)). Appropriate positive human control tissues included placenta for PRR, liver for ACE and ATIIR1, and kidney for ATIIR2.
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