Samples staining and imaging

3.6.1. Immunostaining

3.6.1.1. Immunostaining of cell lines

After a PBS washing, cells where fixed with PFA 4% for 1h, washed again and permeabilized with 0.1 % SDS (sodium dodecyl sulphate) in PBS for 10 minutes. After washing three times, samples are incubated o/n in a humid chamber at 4ºC in the presence of the antibody diluted in 10% NGS (normal goat serum). After a long washing step (1h), samples are incubated in the presence of the secondary antibody conjugated with a fluorophore in BGT buffer (3mg/mL BSA, 10 mM Glycine and 0.25% Triton X-100 in PBS) during 1h at RT in a humid chamber. After another long washing step, the samples are stained with the nuclear marker DAPI (4',6- diamidino-2-phenylindole) at 1 μg/mL (Invitrogen, California, USA), washed again and finally, mounted with Fluoromount-G (Cultek).

3.6.1.2. Immunostaining of retinal cryosections

Frozen samples at -80ºC were tempered first at RT for 10 minutes and then, at 50ºC 30 minutes in a stove. Once dry, samples were rehydrated in PBS and then blocked and permeabilized in BGT buffer for 30 minutes. After washing three times with PBS, samples were incubated o/n in a humid chamber at 4ºC in the presence of the primary antibody mixture diluted in BGT. After three long washing steps (1h), samples were incubated in the presence of the secondary antibody conjugated with a fluorophore in BGT during 1h at RT. After another three long washing steps, the samples were mounted with DABCO with the nuclear staining DAPI at 1 μg/mL.

3.6.1.3. Immunostaining of flat-mounted retinas

Samples were washed three times in PBS and blocked and permeabilized in BGT buffer for 30 minutes. After washing three times, a stronger permeabilization is performed with 1% of triton X- 100 for 90 minutes plus another washing step (three times). Then samples were incubated o/n in a humid chamber at 4ºC in the presence of the primary antibody cocktail diluted in BGT. After three long washing steps (1h), samples were incubated with the secondary antibodies mixture in BGT during 2h at RT. After another long washing step, the samples are mounted with DABCO with DAPI at 1 μg/mL.

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3.6.1.4. Immunostaining of cerebellum cryosections

As samples were stored at -80ºC, they were tempered at RT for 10 minutes and then dried at 50ºC in a stove. Once ready, samples were washed three times in PBS and blocked and permeabilized with a blocking buffer (1% triton X-100, 1% BSA and 3% FBS in PBS) for 2 days at 4ºC. After washing three times, samples are incubated o/n in a humid chamber at 4ºC in the presence of the primary antibody cocktail diluted in the same blocking buffer. After three long washing steps (1h), samples were incubated in the presence of the secondary antibody diluted in the blocking solution during o/n at 4ºC. After another three washing steps, the samples are mounted with DABCO with the nuclear staining DAPI at 1 μg/mL.The analysed areas were the molecular, Purkinje and granular layers.

3.6.1.5. Primary and secondary antibodies for immunofluorescence staining

The proteins detected by immunostaining in the conditions detailed bellow are listed in the Table 3.1 Secondary antibodies are listed in Table 3.2.

Table 3.1. List of the primary antibodies used for the immunostaining procedures commented above, detailing the provider, reference, animal species of obtainment and used dilution.

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3.6.2. Apoptosis labelling by TUNEL staining (dead end fluorimetric system)

The TUNEL staining detects the fragmented DNA of apoptotic cells by incorporating fluorescein- 12-dUTP at the 3’ -OH ends of the DNA using the enzyme terminal deoxynucleotidyl transferase (recombinant), which forms a polymeric tail using the principle of the TUNEL (TdT- mediated dUTP Nick-End Labeling) assay.

The assay was performed in retinal cryosections. Slides were rehydrated in PBS, permeabilized with 2% triton for 1h and blocked with BGT for 30 minutes. After washing, samples were incubated 15 minutes in equilibration buffer (200 mM potassium cacodylate, 25mM Tris-HCl, pH 6.6, 0.2 mM DTT, 2.5 cobalt chloride, 0.25 mg/mL BSA). Once equilibrated, the samples were incubated 1h at 37ºC with the TUNEL reaction mix (2% rTDT enzyme, 10% nucleotide mix, 88% equilibration buffer). The reaction was stopped with 2x SSC (Salt Sodium Citrate).

3.6.2. Photoconvertible fluorescent reporter to measure chaperone-mediated autophagy activity

The pKFERQ-PA-mCherry1 plasmid construction and the establishment of stable cell lines expressing the CMA reporter was performed using lentiviral transfer vectors as described by (Koga et al., 2011b). The reporter is linked to the KFERQ motif of the proteins that carry it and it get photoactivated under UV light exposure. Thus, for the experiments, photoactivation of the

Table 3.2. List of the secondary antibodies used in the immunostaining procedures commented above, detailing the provider, reference, animal species of obtainment and used dilution.

Figure. 3.6. Scheme of the experimental design to monitor CMA activity in cells transfected with pKFERQ-PA-mCherry1 reporter.

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reporter was performed on transfected cells grown on coverslips with a 405/20 nm LED array (Norlux) for 10 min using 50 mW cm-2 light intensity previously to the CMA induction. Hence, only the proteins with the photoactivated reporter will be followed and not those synthesized during the experiment, so we could estimate the CMA activity. More than 90% of the cells were viable after the photoactivation. These experiments were carried out at the laboratory of Dr. Ana Maria Cuervo, Albert Einstein College of Medicine (New York, USA). Depending on the experiment, images were acquired by different microscopes. For eplifluorescence a Zeiss Axioscopp microscope was used and for confocal acquisition, a Leica TCS-SP5-A0BS.

3.6.2. ONL thickness measurement

This technique allows following the degeneration of the photoreceptor nuclear layer in an easy way. Retinal cryosections stained with DAPI and mounted with DABCO were used for the measurement.

3.6.3. Images acquisition

Images were taken with an epifluorescence microscope at 40x of magnification. Six images were taken in total on every section, three at each side of the optic nerve, as disposed in Figure 3.6 at least, 3 sections of every sample are needed for the quantification.

Using ImageJ software, the thickness of ONL and INL was measured at three points in every image (1, 2, 3 in Figure 3.7). Then, we calculated the ratio ONL/INL at each point and the mean in the picture, and, finally, the mean of the three images acquired in each position (A, B, C, D, E, F) in the cryosection. This is repeated in at least three cryosections for each sample.

Figure 3.7. Representation of the measurement of the ONL and INL thickness in a retinal cryosection. Left illustration exemplifies a retinal cryosection and the 6 acquisitions distributed along the retina (A, B, C, D, E, F). Right image shows an example of a retinal cryosection stained with DAPI, showing the three nuclear layers: ONL, INL and GCL. Here, there are represented three example points for the measurements performed in each image (1, 2, 3). ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion nuclear

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The ratio ONL/INL is calculated in order to relativize the value of the ONL to a constant value, as the INL thickness, eliminating errors associated with eye orientation before sectioning and making it comparable parameter between samples.