Diamidine

The immunofluorescent staining of cell nuclei and γH2AX foci was performed according to the following protocol; cells grown on cover slips were washed in PBS and fixed in 4 % paraformaldehyde (in 1xPBS, pH 7.4) for 15 min at room temperature. After three washing steps with PBS, cells were permeabilized using 0.1 % Triton-X 100 (in 1xPBS, pH 7.4) for 15 min at room temperature and then incubated with the blocking reagent (5 % Bovine serum albumin in 1xPBS, pH 7.4) for 45 min. The primary antibody anti-γH2AX (Ab26350, Abcam) was diluted to 1:1000 in 1 % Bovine serum albumin, (in1xPBS pH 7.4) and added to the cells for 2 h at room temperature. After the incubation, cells on cover slips were washed three times in PBS and the fluorescent-labelled secondary antibody diluted 1:500 in the same buffer was added to cells (IgG-Alexa488, Cell Signaling #4408). The samples were stored in the dark at room temperature for 1 h. After washing, the DNA was stained with 49-6-diamidine-2-phenyl indole (DAPI, Invitrogen) diluted to a final concentration 1 μg/ml in the same buffer for 5 min at room temperature. Cells were then washed in PBS and mounted with the anti-fade medium (Vectashield).
Then cells were imaged using a confocal fluorescent laser scanning microscope (FluoView1000, Olympus) with a 60 × oil objective with numerical aperture (N.A.) equal to 1.35. In addition, cells were visualized using a conventional wide-field fluorescent microscope (Keyence BZ-8100E) with a 20× objective with N.A. equal to 0.4. As a result, TIF-images were obtained, where nuclei and foci were detected by blue and green channels, respectively.

Hematoxylin-eosin (H&E) and Masson's trichome staining were performed using the standard procedure for visualizing morphologic details, as reported earlier.^{40 (link),45 (link)} Immunofluorescence staining was performed following reported methods^{17 (link)} to measure myofibroblasts in the corneas using antibody-specific α-smooth muscle actin (α-SMA), a marker for myofibroblasts. Briefly, corneal sections were blocked with 2% bovine serum albumin at room temperature for 30 minutes, followed by incubation with α-SMA mouse monoclonal primary antibody (1:200 dilution, M0851; Dako, Carpentaria, CA, USA) for 90 minutes and then incubated with Alexa-Fluor 488 goat anti-mouse IgG secondary antibody (1:1000 dilution, A11001; Invitrogen, Carlsbad, CA, USA) for 1 hour at room temperature. Appropriate positive and negative controls were included in each immunostaining. Quantification of α-SMA-positive cells was performed in six randomly selected, nonoverlapping, full-thickness central corneal columns, extending from the anterior stromal surface to the posterior stromal surface at 200× and 400× magnification fields.
The toxicity of PEI2-GNPs and BMP7+HGF gene therapy was determined by performing a TUNEL assay (ApopTag; Millipore, Temecula, CA, USA). Corneal sections were fixed in acetone at –20°C for 10 minutes, and a TUNEL assay was performed per the manufacturer's instructions, including suitable positive and negative controls. Rhodamine-conjugated apoptotic cells (red) and 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI)–stained nuclei (blue) were viewed and photographed with a fluorescence microscope (Leica) fitted with a digital camera system (SpotCamRT KE; Diagnostic Instruments, Sterling Heights, MI, USA). DAPI-stained nuclei and TUNEL-positive cells in untreated and treated tissues were quantified at 200× and 400× magnification in six randomly selected nonoverlapping areas, as previously reported.^{17 (link),28 (link)}

At the end of the experiment, we fixed the adipose tissue and liver in 10% formalin for 30 min. After dehydration, we embedded the fixed tissues in the frozen optimal cutting temperature (OCT) compound and paraffin, respectively, and then sectioned them using a Leica Cryostat at 6 mm in thickness. We stained the tissue sections with hematoxylin and eosin (H&E) or Oil-Red-O (Lot: 031M0143V, Sigma) and examined them under a light microscope (Olympus IX51; Olympus, Tokyo, Japan) at ×100 magnification. For immunofluorescence analysis, the paraffin-embedded GWAT sections were incubated with FITC-conjugated mouse F4/80 antibody (Lot: C4801011618354, Tonbo Biosciences, San Diego, CA, USA) for 2 h at 4 °C. After washing with PBS, the slides were counterstained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI, Sigma) for 10 min at room temperature, washed, and then observed at 100× magnification under a fluorescence microscope (Olympus).

Due to an essentiality of nuclear localization for DNA-repair cellular events, the subcellular localization of Rad4A and Rad4B was explored using the green fluorescence protein GFP-tagged fusion protein of each expressed in the wild-type strain B. bassiana ARSEF 2860 (denoted as WT hereafter) as described previously [36 (link)]. For each target gene, the open reading frame (ORF) amplified from the WT cDNA with paired primers (Table S1) was ligated to the 5′-terminus of gfp (U55763) in the linearized pAN52-gfp-bar vector using a one-step cloning kit (Vazyme, Nanjin, China). The constructed vector pAN52-x- gfp-bar (x = rad4A or rad4B) controlled by the endogenous promoter Ptef1 was integrated into the WT genome via a transformation mediated by Agrobacterium. Transgenic colonies were screened via the bar resistance to phosphinothricin (200 μg/mL) and examined under a fluorescence microscope. Based on the desired green signal, a colony selected from those generated by each transformation was incubated for conidiation on SDAY (Sabouraud dextrose agar (4% glucose, 1% peptone and 1.5% agar) plus 1% yeast extract) at the optimal regime of 25 °C and 12 h light:12 h dark. The resultant conidia were suspended in SDBY (agar-free SDAY), followed by a 3-day incubation on a shaking bed (150 rpm) at 25 °C. Hyphal samples taken from the cultures were stained with 4.16 mM DAPI (4′,6′-diamidine-2′-phenylindole dihydrochloride; Sigma-Aldrich, Shanghai, China) and visualized for subcellular localization of Rad4A-GFP or Rad4B-GFP via laser scanning confocal microscopy (LSCM) at the excitation/emission wavelengths of 358/460 and 488/507 nm. For either fusion protein, green fluorescence intensity was assessed from a fixed circular area moving in the cytoplasm and nucleus of each of 15 hyphal cells using ImageJ software (https://imagej.nih.gov/ij/, accessed on 23 January 2023) and used to compute nuclear versus cytoplasmic green fluorescence intensity (N/C-GFI) ratio as its relative accumulation level in the nucleus of each hyphal cell.
Co-localization of either Rad4A or Rad4B with Rad23 was explored to reveal a possible interaction of either with Rad23. Briefly, the rad23 ORF was ligated to the 5′-terminus of mCherry (KC294599) in the vector pAN52-mCherry-sur under the control of Ptef1 [36 (link)]. The new vector was transformed into the respective strains expressing Rad4A-GFP and Rad4B-GFP as aforementioned, followed by the screening of transgenic colonies via sur resistance to chlorimuron ethyl (10 μg/mL). A colony showing desirable red fluorescence was chosen for subcellular co-localization of Rad4A-GFP or Rad4B-GFP with Rad23-mCherry by LSCM at the excitation/emission wavelengths of 488/507 and 561/610 nm.