Alexa fluor 488 conjugated goat anti rabbit immunoglobulin g

To determine whether cell death in the organ of Corti occurred via apoptosis, we performed immunohistochemistry for active caspase-3 and TUNEL assay. After fixation, specimens were permeabilized with 0.1% Triton X-100 in PBS (PBS-Tx) for 30 min at RT and blocked with 5% normal goat serum for 1 h at RT. The specimens were incubated with anti-active caspase-3 (1:1000; Cell Signaling Technology, Beverly, MA, USA) diluted in blocking solution overnight. After three rinses with PBS, samples were labeled with Alexa Fluor^® 488-conjugated goat anti-rabbit immunoglobulin G (1:1000; Invitrogen, La Jolla, CA, USA) diluted in blocking solution for 1 h at RT. F-actin was labeled with Alexa Fluor^® 555-conjugated phalloidin in PBS-Tx for 1 h at RT.
To detect DNA fragmentation, a marker of apoptotic cell death, we used the TUNEL assay kit (Promega, Madison, WI, USA) following the manufacturer’s protocol. Specimens were fixed with 4% paraformaldehyde in PBS for 15 min at RT and rinsed with PBS. And then, they were permeabilized with 0.1% PBS-Tx in 0.1% sodium citrate for 30 min at 37 °C and stained with TUNEL working solution for 30 min at 37 °C. F-actin was labeled with Alexa Fluor 555-conjugated phalloidin in PBS-Tx for 3 h at RT in the dark. Specimens were mounted on glass slides using Fluoromount and visualized using a Zeiss Axio Imager A2 fluorescence microscope.

Nuclear NRF2 translocation was determined by the immunofluorescence assay as previously described by Zhang et al. [35 (link)], with minor modifications. Briefly, IPEC-1 cells were fixed in 4% paraformaldehyde solution in 0.1 M PBS at room temperature for 30 min after washing twice with PBS. The fixed cells were further permeabilized with 0.5% Triton X-100 for an additional 30 min. Following the washing with PBS, cells were incubated with the blocking buffer (5% bovine serum albumin in PBS) for 1 h at 4 °C before incubation with a rabbit anti-NRF2 antibody (1: 100, Proteintech Group, Inc., Chicago, IL, USA) in PBS at 4 °C overnight. After washing with PBS with 0.1% Tween 20, cells were incubated with a secondary antibody, Alexa Fluor 488-conjugated goat anti-rabbit immunoglobulin G (1: 100, Invitrogen, Carlsbad, CA, USA), in dark conditions for 1 h at 4 °C. The 5 µg/mL 4,6-diamidino-2-phenylindole was adopted for nuclear staining. Images were visualized with an Eclipse 80i inverted fluorescence microscope (Nikon Instruments, Tokyo, Japan), and nuclear NRF2 fluorescence intensity was calculated using the Image J software (National Institute of Health, Bethesda, MD, USA) and normalized against control cells.

Tissues were fixed in paraformaldehyde overnight, embedded in paraffin, and cut at a thickness of 5 μm and then stained with hematoxylin and eosin. We used the methods of Shoshani [23 (link)] and Yu [24 ] to evaluate the histologic parameters, such as cell integrity, tissue inflammation, presence of cysts/vacuoles, and the extent of fibrosis. Each parameter was scored as 0 = absence, 1 = minimal presence, 2 = minimal to moderate presence, 3 = moderate presence, 4 = moderate to extensive presence, and 5 = extensive presence. The scoring was performed independently by 3 authors who were unaware of the grouping.
For immunofluorescent staining, tissue sections were incubated with primary antibody against Perilipin (Proteintech, #15294-1-AP, China, 1:200) diluted in blocking solution overnight at 4 °C. After incubation with Alexa Fluor 488–conjugated goat anti-rabbit immunoglobulin G (Invitrogen, #A-21206, USA, 1:500), nuclei were stained with 4′,6-diamidino-2-phenylindole (Southern Biotech, USA). ImageJ software was used for quantitative analysis. The image analysis was refered to the website (https://imagej.net/imaging/image-intensity-processing) and the method of Keskin [25 (link)].

For immunofluorescent staining, tissue sections were incubated with primary antibody against Perilipin (Abcam, ab172907, 1:200) diluted in blocking solution overnight at 4°C. After incubation with Alexa Fluor 488–conjugated goat antirabbit immunoglobulin G (Invitrogen, USA, 1:500), nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (SouthernBiotech, USA). Image‐Pro Plus 6.0 software was used for quantitative analysis.