Fluorescent microscopy

HRECs were plated at 1 × 10⁵ in 8-well chamber slides (Sigma-Aldrich Chemical Corp., St. Louis, MO, USA) and treated with and without Hcy (20, 50, or 100 μM) for 24 hr. Cells were then fixed with 4% formalin for 10 min, washed with PBS, and blocked with Power Block (BioGenex, Fremont, CA, Ca. #BS-1310–25) for 1 hr. Thereafter, the cells were incubated at 4 °C overnight with Antibodies for ZO-1(Abcam, Cambridge, Massachusetts, USA, Cat.# ab59720), occludin (Invitrogen, Eugene, Oregon, USA, Mouse monoclonal Cat.# 33–1500), claudin-5 (Invitrogen, Eugene, Oregon, USA, Rabbit Polyclonal Cat.# 34–1600), anti-GSH-1 (Santa Cruz, Dallas, Texas, USA. Cat.# sc-292189), anti-Nrf2 (Abcam, ab137550), anti CD31 (Novus Biologicals, NB100–2284) and anti α-smooth muscle actin (Abcam, ab5694). After primary antibody treatment, cells were then washed 3 times with PBS containing 0.3% Triton-X and incubated with appropriate secondary antibodies (Alexafluor and Texas red avidin, Invitrogen). Slides were cover-slipped using Fluoroshield containing DAPI (Sigma-Aldrich) as a counter stain. Images were captured by fluorescent microscopy (Carl Zeiss, Göttingen, Germany).

Comet assays or single cell gel electrophoresis were performed as described [21 (link)]. Cells were gently collected avoiding exposure to light. All incubations were performed in the dark at 4 °C. Briefly, 80000 cells were embedded in prewarmed (37 °C) 0.5% low melting point agarose (Invitrogen, 15517–014) and pipetted in slides (previously prepared and covered with one layer of 0.5% normal melting point agarose; Pronadisa, 8016). After solidification, another layer of 0.5% low melting point agarose was added to the slides. Samples were incubated for 1 h in cold lysis solution (10% DMSO, 1% Triton X-100) and 89% lysis buffer (pH 10, NaCl 2.5 M, EDTA 100 mM, Tris 10 mM, NaOH 200 mM). Then, electrophoresis was carried out at 25 V and 4 °C for 20 min. Finally, samples were washed in Tris 400 nM pH 7.5 and fixed in absolute ethanol. After DNA staining by RedSafe (1:500; InTron, 21141), samples were visualised under a fluorescent microscopy (Zeiss, Germany). DNA damage was quantitated by measuring the comet tail length in pixels by the ZEN 2012 programme (Zeiss).

Kidneys were embedded in OCT compound (Tissue-Tek, Torrance, CA) and snap-frozen. The freshly cut renal cryostat sections (10μm) were used for in situ reactive oxygen species (ROS) measurement. Dihydroethidine hydrochloride (DHE) (5μM, Molecular Probes, Grand Island, NY) was topically applied to the sections for 30 min at 37°C to reveal presence of ROS as red fluorescence by fluorescent microscopy (Carl ZEISS, Thornwood, NY). Quantitative analysis was performed using Image J software.

Neutral comet assay was performed using the Trevigen CometAssay Reagent kit as per manufacturer instructions. Cells were visualized using fluorescent microscopy (Carl Zeiss). Images were analyzed using Comet Assay IV System (Perceptive Instruments Ltd, UK).

Mice were perfused with 10 ml 1× PBS and 20 ml 4% paraformaldehyde (PFA). After extraction, the brains were post-fixed with 4% PFA for 24 hr. Tissues were then transferred to 15% sucrose solution for 24 hr, followed by immersion in 30% sucrose solution for another 24 hr. After that, the brains were then embedded in the Optimal cutting temperature compound and cut into 5 μm thick sections. For the immunofluorescence staining, sections were dried at 37°C for 10 min, then wash with 1× PBS at RT for 5 min. Sections were incubated with 5% BSA at RT for 30 min, followed by primary antibody staining at 4°C for overnight (mouse anti-82E1 [1:200], hamster anti-CD11c [1:100], and rabbit anti-Iba-1 [1:200]), three times washings with 1× PBS and an incubation with the correspondent secondary antibodies (Goad anti-hamster FITC [1:200], Donkey anti-rabbit Alexa Fluor 594 [1:200], and Goat anti-mouse APC [1:200]). Stained sections were then incubated with DAPI for 10 min and mounted with fluorescence mounting medium and visualized by fluorescent microscopy (Zeiss). Images were post-processed by Zen software.