*2.5. Immunofluorescence*

Rat eyes were embedded in OCT mounting medium (Tissue-Tek), frozen on dry ice and then cryostat sectioned. A 4% paraformaldehyde fixative was applied to the slides for 10 min. Slides were incubated overnight at 4 ◦C with one of the following primary antibodies: Rabbit anti-HDAC6 (Lifespan Biosciences, Seattle, WA, USA) and mouse anti-phosphorylated form of H2A histone family member X ( γH2AX) (Cell Signaling Technology, Danvers, MA, USA). Slides were washed three times with 0.1% Triton X-100 in 0.1 M PBS (pH 7.4) followed by a 1-h incubation with one of the following secondary antibodies, all purchased from Molecular Probes-Life Technologies (Grand Island, NY, USA): goa<sup>t</sup> anti-rabbit IgG-conjugated Alexa Fluor 488, goa<sup>t</sup> anti-mouse IgG-conjugated Alexa Fluor 488. Slides were mounted using Fluoroshield mounting medium containing 4-,6-diamidino-2-phenylindole (DAPI) to visualize nuclei (Sigma-Aldrich). Sections were examined for epifluorescence using a Zeiss Axioplan-2 fluorescence microscope (Carl Zeiss) equipped with the Axiovision program (version 4.7; Carl Zeiss).

#### *2.6. Assessment of HDAC6 Activity*

HDAC6 activity was assessed in both rat and human cell samples using a commercially available fluorimetric assay kit (Biovision, Milpitas, CA, USA), employing a synthetic acetylated-peptide substrate resulting in the release of an AFC fluorophore, which can be detected and quantified with Ex/Em, 380/490 nm at 37 ◦C.

#### *2.7. Assessment of Thioredoxin-1 Activity*

Thioredoxin activity fluorescent assay kit (Cayman Chemical, Ann Arbor, MI, USA) was used to assess the activity of thioredoxin-1 (Trx-1) in rat retinal extracts and HuREC lysates. This assay measures the ability of endogenous Trx-1 to reduce the disulfides of fluorescently labeled insulin. The resulting fluorescent signal, measured at Ex/Em, 520/545 nm, is a direct measurement of Trx-1 reducing activity.
