Darkness

The IHC images used were stained with DAB and hematoxylin. The result of color deconvolution leads to the production of three images, namely, DAB, hematoxylin and a complimentary image. In a previous study, we reported development of an ImageJ compatible plugin for analyzing cytoplasmic staining pattern by assigning a histogram profile for the deconvoluted DAB image [21] . Now, within the scope of the current plugin development, we envisioned automating the whole process by integrating deconvolution, histogram profiling and scoring by a simple choice of the program menu. Additionally, it integrates methods with a wider scope of analyzing various marker proteins displaying cytoplasmic or nuclear staining patterns (Table 1).
In digital image analysis, the pixel intensity values for any color range from 0 to 255, wherein, 0 represents the darkest shade of the color and 255 represent the lightest shade of the color as standard. A total of 1703 images were analyzed independently with the help of two expert pathologists and were assigned a score as high positive (3+), positive (2+), low positive (1+) and negative (0). In the current method development, the next step was assigning a histogram profile which is a plot between the intensity values of the pixels (X axis) vs. the number of pixels representing the intensity (Y axis). Keeping in view the standard grading procedure, the histogram profile was divided into 4 zones, viz. high positive, positive, low positive and negative. These four zones were equally divided on the pixel color intensity bar (as indicated in Figure S1A).
The zones were visually identified by using the threshold feature of Image menu of the ImageJ program [22] (link). To begin with, the intensity values were grouped into bands of 10 and the corresponding regions in the image were confirmed by using the threshold feature. Thus initially all the intensities from 0 to 10 were turned red on an image with a known pathological score of high positive. Then, in addition to the previous band, the next band, from 11 to 20 were turned red and the same was continued until all the pixels of the brown shades were assigned a threshold and the range for the high positive zone was determined. Similarly, zone containing the lightest color shade of pixel intensities was also determined using an image with a known pathological score of negative. This was because once the highest intensity (high positive) and the least intensity (negative) zones were determined, it would help towards the better determination of the size of the intermediate (positive and low positive) zones. It was found that the region between 0 and 60 contained pixels of the high positive stained images. Similar was noted on samples with known pathological lower scores to optimize the correct range. The process was repeated for at least 70 images of same intensity of the color shade. The intensity range for the positive zone was found to be ranging from 61 to 120; 121 to 180 for the low positive zone; and 181 to 235 for the negative zone, respectively (Figure S1). It was determined that the pixels with intensity values ranging from 235–255 predominantly represent fatty tissues which are occasionally present but do not typically contribute to pathological scoring and were therefore excluded from the score determination zones. The intensity ranges determined visually were confirmed by plotting a histogram using Microsoft Excel (data not shown).

The One-step TUNEL Apoptosis Assay Kit (Beyotime, Beijing, China) was used to perform the TUNEL assay (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay) for 8-μm-thick corneal cryosections. Briefly, cryosections of the cornea samples were fixed with 4% paraformaldehyde for 60 min, and then washed thrice with PBS, followed by the addition of 0.3% Triton X-100 and incubation at room temperature for 5 min. Next, for the TUNEL staining, the TUNEL test solution was prepared according to the manufacturer’s instructions. To each sample, 50 μL of TUNEL solution was added and this incubated at 37 °C for 60 min in darkness. After rinsing three times with PBS for corneas, 4ʹ,6-diamidino-2-phenylindole (DAPI) was added to counterstain and seal each cryosection sample. Three cryosections from each rat’s cornea were analyzed and photographed by confocal laser scanning microscopy (CLSM; Zeiss LSM 800, Germany).

25 µg of purified AGA, GUSB CTSD, and GAA were dissolved in 50 mM ammonium bicarbonate (AmBic) buffer (pH 7.4) and further reduced with 10 mM dithiothreitol (DTT) at 60°C for 45 min on shaker, followed by alkylation with 20 mM iodoacetamide (IAA) at 25°C for 30 min in darkness. AGA, GUSB, CTSD were subjected to proteolytic digestion with chymotrypsin (1:40 enzyme-substrate ratio), while GAA was digested in gel with trypsin (1:25 enzyme-substrate ratio) after SDS-PAGE separation. The reaction was quenched with 1 µL trifluoroacetic acid (TFA) and the digested sample was desalted by custom-made modified StageTip colums with three layers of C18 and two layers of C8 membrane (3 M Empore disks, Sigma-Aldrich). Samples were eluted with two steps of 50 µL 50% methanol in 0.1% formic acid. Final sample was aliqoted in two equal parts. The first aliquot was placed into a glass insert (Agilent), dried completely in SpeedVac (Eppendorf) and further re-dissolved in 50 µL 0.1% formic acid (FA) and submitted for nLC-MS analysis. The second aliqout was placed inside an Eppendorf tube, dried completely using SpeedVac, and then re-dissolved in 50 µL of 50 mM AmBic buffer (pH 7.4) and incubated with PNGase F (1U per sample) for 12 h with shaking at 37°C. Samples treated with PNGase F were desalted and dried using the same methods mentioned above for the first aliqout and submitted for nLC-MS/MS analysis.