Inverted fluorescent microscope

H&E staining was performed by the conventional staining method. Briefly, with about 4 min of hematoxylin staining followed by 0.5% eosin staining for 3 min. Images were acquired by an inverted fluorescent microscope (ZEISS, Germany) with 20× objective lens (200× magnification), and the skeletal muscle fiber diameter was measured by Image J software (National Institutes of Health, Bethesda, MD, USA).

Forty-eight hours after transfection with the RNA oligonucleotides, cells in 6-well plates were treated with 50 μM EdU (RiboBio, Guangzhou, China) in culture medium for 2 h, and then fixed with 4% paraformaldehyde for 20 min at room temperature. After washing twice with cold phosphate-buffed saline (PBS), six random fields were selected and photographed under an inverted fluorescent microscope (Carl-Zeiss, Berlin, Germany).

Retinas were isolated via dissection away from surrounding tissues and then washed in PBS. Fluorescence was confirmed via live imaging under an inverted fluorescent microscope (Zeiss). Retinas were then incubated on a nutator in Papain and DNase I for 10 min at 37 °C. Retinas were then triturated to generate a single-cell suspension and transferred to a tube containing an equal volume of Ovomucoid. The suspension was then spun down at 300 g for 10 min at 4 °C. Cells were resuspended in a solution of 100:1:1 Neurobasal:DNase:Ovomucoid, and passed through a 35 μm filter before sorting using a BD FACSAria III Cell Sorter (BD Biosciences). Gating was performed to isolate intact cells from debris and to isolate positive fluorescent glial or progenitor cells. Progenitor cells were isolated from SOX2 + Amacrine cells by removing the higher fluorescent neuronal population. Positive fractions containing fluorescently labelled MG were then spun down at 300 g and resuspended for the appropriate assay.

We collected spermic urine for sperm count and viability estimates by firmly grasping each frog by the front legs and holding it over a Petri dish until it urinated (usually 5–10 s). We collected the sample from the Petri dish using a sterile 100 μl pipette and used approximately half the sample for sperm count analyses and placed the remaining sample in a 1.5 ml microcentrifuge tube for sperm-viability analysis.
Sperm count was conducted using a hemocytometer. We counted each sample twice and used the mean in analyses. Our measures were highly repeatable (repeatability SPSS 20.0; r = 0.95; [21 (link)]). To determine sperm viability (live sperm/total sperm counted), we used SYBR-14 and propidium iodide, a staining method commonly used for amphibians (e.g., [10 (link), 13 , 22 (link), 23 ]). Briefly, we homogenized samples with 5 μl of a 1:50 dilution of SYBR-14 (Invitrogen L-7011, Canoga Park, CA) and incubated for 7 min; we then incubated with 2 μl propidium iodide for 7 min. Both incubations were done in the dark. We analyzed stained samples using fluorescent microscopy under 100× magnification (Zeiss inverted fluorescent microscope, 2012, Dublin, CA). We counted no fewer than 20 sperm per sample (baseline number for examining viability of sperm in amphibians; [24 (link)]); samples with fewer than 20 sperm were not included in viability analyses.

PDAC cells were plated on coverslips and allowed to adhere overnight and expose to BD (2.5 µM) for 24 h. Then the cells were fixed with 4% paraformaldehyde/PBS, blocked with 5% BSA in PBS and incubated with Nrf2 primary antibody (1:100; Santa Cruz #sc-365949) in PBS containing 3% BSA, followed by Cy3-labeled secondary antibody (Abcam, United Kingdom). Nuclei were stained with DAPI (Santa Cruz, Texas, USA). Images were visualized using an inverted fluorescent microscope (Carl Zeiss, Germany).

Sections of aortic root or liver from mice and cells samples were blocked in 1% BSA containing 1% goat serum for 60 min. The slides were incubated with primary antibodies diluted by 1% goat serum overnight at 4 ℃. After incubation with Alexa-Fluor 555-conjugated goat anti-rabbit IgG antibody for 1 h at room temperature. Meanwhile, nuclei were stained with DAPI (blue) and then observed with an inverted fluorescent microscope (Carl Zeiss, Oberkochen, Germany). Calcification and expression of smooth muscle α-actin (α-SMA), ABCA1, ABCG1 and LC3 protein (red) in lesion areas or liver sections were determined by immunofluorescent staining with aortic root cross section or liver section. The colocalization of GFP-MAP1LC3B/LC3B (green) and RFP-LC3B (red) or bodipy (green) and LAMP2 (red) in AcLDL-treated peritoneal macrophages were analyzed by immunofluorescent staining.