Mouse anti vimentin

As previously described,²⁷ (link) mouse eyeballs were fixed in 4% paraformaldehyde (PFA) at 4°C overnight and cryoprotected in 20% sucrose solution, followed by embedding in optimal cutting temperature compound (Sakura, Torrance, CA, USA). Frozen retinal sections (10 µm) were collected and incubated in a blocking buffer (1% BSA, 0.3% Triton X-100 [Sigma-Aldrich, St. Louis, MO, USA] in PBS) for 1 hour at room temperature, followed by incubation with primary antibodies at 4°C overnight. After three rinses with PBS, the sections were incubated with a secondary antibody at room temperature for 1 to 2 hours. Primary antibodies used included rabbit anti-Ki67 (1:100; Abcam, Cambridge, UK), mouse anti-bFGF receptor (1:200; Millipore, Burlington, MA, USA), rabbit anti-recoverin (1:1000; Millipore), mouse anti-cellular retinaldehyde-binding protein (anti-CRALBP; Abcam), rabbit anti-B-opsin (1:250; Millipore), rabbit anti-R/G-opsin (1:250; Millipore), rabbit anti-glutamine synthetase (GS) (1:500; BD Biosciences, San Jose, CA, USA), and mouse anti-vimentin (1:1000; Sigma-Aldrich). All secondary antibodies (Alexa Fluor 488 anti-rabbit, Alexa Fluor 488 anti-mouse, Cy2 and Cy3 anti-rabbit/mouse) were from Jackson ImmunoResearch Laboratories and were used at 1:400 dilution. Sections were mounted with mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; Abcam).

For immunofluorescence, tumor cells were obtained as described in Rowald et al. (2016) (link). Cells were cultured on coverslips coated with Collagen I (Cultrex, 344.020-01). After 24 h, cells were fixed with 4% PFA for 10 min at room temperature or with a methanol/acetone mix for 10 min at −20°C and permeabilized with 0.2% Triton X-100 PBS for 10 min. Cells were treated with blocking buffer containing 5% donkey serum, 1% BSA, and 0.2% Triton X-100 in PBS for 1 h at RT. The following primary antibodies were used: rabbit anti-LC3 (1:100, Sigma, L7543), rabbit anti-Fibronectin (1:40, Abcam, ab23750), mouse anti-Ataxin1 (1:50, Santa Cruz, sc-365343), mouse anti-Myosin X (1:50, Santa Cruz, sc-166720), mouse anti-RhoA/RhoC (1:100, Thermo Scientific, 1B3-4A10), mouse anti-Vimentin (1:50, Sigma, V2258), and rabbit anti-AKT3 (1:800, Cell Signaling, E1Z3W). Donkey anti-mouse Alexa 488 and anti-rabbit Alexa 568 secondary antibodies were used (1:500, Invitrogen), and the DNA was stained with DAPI. Images were acquired with the Leica LAS 4.5 software on a Leica SP5 confocal microscope. Quantification of LC3 fluorescence intensity was performed with ImageJ software.

Immunophenotyping of the GS-1 cell line was carried out at the 30th passage following the method described previously [10 (link)] with some modifications.
Briefly, the cells were grown on coverslips in six-well plates for 24 hr and then fixed with 4% paraformaldehyde at 4°C for 1 hr and permeabilized with 0.2% Triton-X 100 at room temperature
for 15 min. After blocking with 2% bovine serum albumin (BSA) in PBS at room temperature for 2 hr, the cells were then incubated with four different primary antibodies of
mouse-anti-cytokeratin, mouse-anti-vimentin, mouse-anti-fibronectin and rabbit-anti-desmin (Sigma-Aldrich Co., St. Louis, MO, U.S.A.) at a dilution of 1:200 at room temperature for 2 hr to
characterize the cell line. Subsequently, the cells were rinsed three times with PBS and were incubated with the appropriate secondary antibodies of goat-anti-mouse FITC-conjugated or
goat-anti-rabbit FITC-conjugated (Sigma-Aldrich) at a dilution of 1:320 in PBS containing 1% BSA at room temperature for 1 hr. In control coverslips, only PBS with 1% BSA was used in place
of the primary antibodies. Diaminophenylindole at a final concentration of 1 µg/ml was used to stain DNA in nuclei. All samples were observed with a Carl
Zeiss fluorescence microscope.