Histochoice cleaning agent

To determine the area and volume of spared tissue, we followed a modified method of eriochrome cyanine (EC) staining from Rabchevsky and colleagues [38 (link)] and described in detail by Reigada, 2022 [39 (link)]. Briefly, sections were sequentially stained with EC followed by counter-staining with 5% iron alum and borax-ferricyanide solutions. After dehydration with increasing concentrations of ethanol and Histochoice Cleaning Agent (Sigma-Aldrich), the samples were mounted using DPX (Sigma-Aldrich). EC stained white matter myelin and allowed us to differentiate spared white matter from grey matter or damaged tissue. The area and volume of spared white matter were estimated through the stereological analysis of sections comprising 1 cm around the epicentre (200 µm between sections), using Cavalliery’s method in an Olympus BX61microscope (Olympus) equipped with a motorised stage coupled to a computer running the stereology software Olympus VIS system composed by the Visiopharm Integrator System (VIS; Visiopharm, Horsholm, Denmark) software and the NewCast module for stereology acquisition and image analysis. We randomly overlayed a 2D grid of crosses on top of each section image (20,000 µm² per cross) and the number of hitting crosses was used to obtain an unbiased estimate of the areas. The white matter area was relativised to the total area of each section.

Formalin-fixed paraffin-embedded kidney specimens that were obtained from Wistar control and Wistar STZ rats were deparaffined with Histochoice Cleaning Agent (Sigma Aldrich) and ethanol and hydrated in water. Epitopes were retrieved by incubating the specimens in sodium citrate buffer (10 mM, pH 6.0) at 97 °C for 20 min. The specimens were blocked with 5% bovine serum albumin (BSA) for 1 h. Bovine serum albumin was then removed from the slides, and primary antibodies (diluted in 5% BSA) were added to the specimens, followed by overnight incubation at 4 °C (Table 2). The next day, the specimens were incubated with secondary antibodies (Cell Signaling Technology, Danvers, MA, USA, catalog no. 8114S or 8125S) at room temperature for 30 min. The slides were then incubated for 2 min with Signal Stain DAB substrate (Cell Signaling Technology). The specimens were counterstained with hematoxylin for 5 s and dehydrated with ethanol and Histochoice. Cells were imaged using a Nikon Eclipse Ti microscope. Quantification of the amount of stained proteins was performed using ImageJ 1.52a software (National Institutes of Health).