*2.3. Histopathological Investigation*

All surgically peeled membrane samples were embedded in the optimal cutting temperature compound (OCT)-medium (Leica Biosystems, Nussloch GmbH, Germany) and stored at −20 ◦C initially for slow freezing. After freezing, sections of 4 μm thickness were prepared using cryostat (Leica CM1950 Clinical Cryostat-Leica Biosystems, Nussloch GmbH, Germany) for each subject. The sections were stained with hematoxylin and eosin (H&E) dyes and images were taken at 4× and 10× with an inverted microscope (Olympus Corporation, Shinjuku, Tokyo, Japan).

#### *2.4. Ultrastructural Evaluations of Membranes Using Transmission Electron Microscopy*

The surgically excised membranes were stored in 3% glutaraldehyde at room temperature for 24 h followed by the standard procedures [11,12] for sample preparation. Ultra-thin (70 nm) sections were made with a diamond knife on ultramicrotome (Leica Ultra cut UCT-GA-D/E-1/00), mounted on gold grids and stained with saturated aqueous uranyl acetate (UA) and counter stained with Reynolds lead citrate (LC) and viewed under TEM (Model: CM10,Philips, North Worcester, MA, USA).

#### *2.5. Cellular Characterization Using Immunohistochemistry*

The tissue sections were processed for immunohistochemistry using cell type specific primary antibodies including rabbit anti-glial fibrillary acid protein antibody (GFAP) (glial marker, 1:400, Z0334, Dako, Glostrup, Denmark), rabbit anti-ALDH1L1 (astrocytes, 1:500, ab190298, Abcam, Cambridge, MA, USA), mouse anti-cellular retinaldehyde-binding protein (CRALBP) (retinal astrocytes, 1:80, ab15051, Abcam), rat anti-F4/80 (macrophages, 1:30, ab16911, Abcam,), and rabbit anti-oxidation resistance 1 (Oxidative stress, 1:100, HPA027395,Sigma, St. Louis, MO, USA), against OXR1; a protein that controls the resistance of neuronal cells to oxidative stress. After sectioning the slides were stored at −20 ◦C. For the experiment, slides were taken out from the −20 ◦C and kept at room temperature for 30 min to bring them to room temperature. Next, the slides were dipped in fixative (chilled methanol: acetone (95:5) solution) for 15 min and dired for 30 min at room temperature. The slides were then washed three times with 1× PBS. 1% BSA (Himedia, Lab Pvt. Ltd., Mumbai, Maharashtra, India) and incubated with blocking bu ffer containing 0.1%-Tween20 (Fisher Scientific, Guldensporenpark, Merelbeke, Belgium) for 2 h. The sections were then incubated with the primary antibody overnight. The primary antibodies were diluted in 0.1% blocking bu ffer. After overnight incubation, the slides were washed three times with 1X PBS, for 5 min each, followed by secondary antibody incubation at a dilution 1:300 (Alexa Fluor ® 488 Goat Anti-Rabbit IgG, (A-11008, Invitrogen, Waltham, MA, USA) and Alexa Fluor® 488 Goat Anti-Mouse IgG, (ab150117, Abcam). Negative control IHC was done to test the specificity of antibody and to identify false positive results shown in Figure S1. Details of the antibodies are provided in Table S1.

#### *2.6. Imaging and Image Analysis*

The digital inverted microscope (EVOS FL Cell Imaging System, Life Technology, Paisley, UK) was used for the imaging at X200 final magnification. Further, total number of cells based on DAPI staining and the total number of positively stained cells for each marker were counted on individual sections corresponding to different pathological conditions. Average and standard deviation of the mean number of cells for each marker from specimens (*n* = 3) in different pathological conditions were calculated. Percentage of OXR1 positive cells was calculated using: (total number of OXR1 positive cells divided by total number of DAPI stained cells) × 100.

#### *2.7. Targeted Gene Expression Profiling by qRT-PCR*

Total RNA was isolated from membranes using PureLink™ RNA Mini Kit (Invitrogen™, catalog no. 12183018A) following the manuals as instructed. RNA was converted to cDNA using verso cDNA synthesis kit (Thermo Scientific™, Waltham, MA, USA, catalog no. AB1453B). A reaction mixture of volume 10μL was made using iTaq™ Universal SYBR® Green Supermix (BIO-RAD, Hercules, CA, USA, catalog no. 38220090), 200nM of primer and cDNA and further, subjected to Applied Biosystems 7900 HT system. A relative measurement of the concentration of target gene (CT) was calculated by using software SDS 2.4 (Applied Biosystems, Foster City, CA, USA). Differential gene expression was analyzed using the <sup>2</sup>−ΔΔ*C*T method. Statistical analyses were performed using the <sup>2</sup>−ΔΔ*C*T ± SEM. β*-actin*; a housekeeping gene was used as a normalizing control. The primer sequences used for semi-quantitative real time PCRs are given in the Table S2.
