Immunostaining and quantification of TFEB nuclear translocation using paraffin-embedded human retinal sections

Human control and AMD donor paraffin-embedded eye globes were subjected to dewaxing using three washes of fresh xylene and rehydration using a gradient of ethanol followed by antigen retrieval as explained previously^22,24. The slides were then washed with PBS/0.1% Tween 20, twice for 10 min each, and then subjected to permeabilization and blocking steps as previously demonstrated^22,24. The slides were incubated with TFEB primary antibody (A303-673A, Bethyl Laboratories) in primary antibody buffer (1% Tween 20 + 0.5% BSA in PBS) at a dilution of 1:100 and incubated in a moist chamber overnight at 4 °C. The slides were washed with wash buffer three times. A secondary antibody cocktail was prepared containing Anti-Rabbit Alexa fluor 647 (1:200) in secondary antibody buffer (0.1% Tween 20 + 0.5% BSA in PBS) also containing Hoechst (1:2000) and was added to each slide covering the sections completely and incubated in dark for 2 h at RT. Post-staining the slides were washed with 1× PBS, 5 times, for 15 min each and then mounted with a coverslip using a DAKO mounting media until imaging in a confocal microscope^22,24. Slides were imaged using VS200 Olympus Slide Scanner with 40× objective (UPlanXApo NA 0.95) in spectral ranges: DAPI (ex. 378/52, em. 432/36), Cy3 (ex. 554/23, em. 595/31), Cy5 (ex. 635/18, em. 680/42) and Cy7 (ex. 735/28, em. 809/81) with ORCA Fusion BT digital CMOS camera. Cy7 channel was used to detect the RPE layer by thresholding followed by morphological operations. Individual nuclei were detected using Cellpose⁵⁶ generalist segmentation algorithm (model ‘cyto’) in the DAPI channel. TFEB-specific signal was calculated as a difference between Cy5 and Cy3 channels. Individual foci were detected using the Difference of Gaussian algorithm (scikit-image⁵⁷). A nucleus was considered positive if more than two foci were detected within its area.