Nis elements basic research imaging software

A 4–5 mm section was cut transversely from the center of the right kidney, and fixed in 10% neutral buffered formalin (NBF) before being microwaved (55°C, 250 watts) for 5 min. Sections were left in 10% NBF at room temperature overnight before being transferred to 70% ethanol, dehydrated by passage through 90% and 100% ethanol, cleared in xylene and embedded in paraffin wax. Sections were cut at 3 µm and stained with either picrosirius red (SR) with light green counterstain or periodic acid‐Schiff (PAS), followed by mounting in DPX. Stained sections were viewed using a Zeiss Axioplan Microscope (Zeiss), and images of representative regions were recorded using a Nikon microscope camera (DS‐Ri2, Nikon) with Nikon proprietary software (NIS Elements Basic Research imaging software, Nikon).
The extent of renal cortical fibrosis was quantitatively assessed using SR by capturing a minimum of 10 serial, nonoverlapping regions (×50 magnification; an area of 0.34 mm²), free of blood vessels, across the mid renal cortex from each animal. The extent of fibrotic tissue was quantified by applying trained pixel classifier software (NIS Elements Basic Research imaging software, Nikon) to each region and expressed as a percentage of each selected region.

Fixation and quantification of the kidney followed methods previously described [56 (link)]. In brief, a transverse 4-5mm section was taken from the centre of the right kidney, fixed in 10% NBF and microwaved (55°C, 250 watts) for 5 minutes. Sections were left in 10% NBF at room temperature overnight before being dehydrated by passage through alcohols and embedding in paraffin wax. Kidney tissue was cut at 3μm and stained with either picrosirius red (SR) or periodic acid-Schiff (PAS), followed by mounting in DPX. Stained sections were viewed using a Zeiss Axioplan Microscope (Zeiss, Germany), and images of representative regions were recorded using a Nikon microscope camera (DS-Ri2, Nikon, USA) and Nikon proprietary software (NIS Elements Basic Research imaging software, Nikon, USA).
Renal cortical fibrosis was quantitatively assessed using SR by capturing a minimum of 10 serial, non-overlapping regions (x50 magnification), containing no blood vessels, across the mid renal cortex from each animal. The extent of fibrotic tissue was quantified by applying trained pixel classifier software (NIS Elements Basic Research imaging software, Nikon, Japan) to each region and expressed as a percentage.

The brain was embedded in a paraffin block and sliced in 5 μm sections. For immunohistochemistry (IHC) staining, the paraffin sections were stained with mouse monoclonal antibody (Santa Cruz Biotechnology, Texas) for detecting the expressions of 4-HNE (1:300 dilution), Ki-67 (1:500 dilution), GFAP (1:500 dilution) and Iba-1 (1:500 dilution) according to the manufacturer’s instructions. Images from slides were viewed at 20× and 40× magnifications under Nikon H600L microscope, camera DS Fi2 and analysed using NIS Elements Basic Research imaging software (Nikon Corp, Japan). The results were scored using Immuno Reactive Score (IRS) as suggested by Remmele and Stegner (10 (link)).