Anti-p62/SQSTM1 antibody produced in rabbit

Protocol tips

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	Dilute primary Ab at 1:5000 and incubate overnight

Protocol tips
Dilute primary Ab at 1:5000 and incubate overnight

Publication protocol

For fluorescence microscopy, HeLa cells were transfected with GFP-tagged LC3 using Lipofectamine 2000. Dot formation by GFP-LC3 was detected with a fluorescence microscope (Olympus IX81) as previously described [45]. Transfected cells with five or more puncta were considered to have accumulated autophagosomes. A total of 100 transfected cells were examined per well, in triplicate, from three independent experiments.

For immunofluorescence microscopy, cells were plated on coverslips (NEST, 801008) 24–48 h before immunostaining. Cells were washed twice with ice-cold PBS and were fixed in 4% paraformaldehyde (PFA) for 30 min, permeabilized in 0.2% Triton X-100 for 15 min and incubated for 60 min in 3% Bovine Serum Albumin (BSA). Cells were then incubated with primary antibody overnight followed by a secondary antibody in PBS containing 3% BSA. Nuclei were stained with 5 μg/mL DAPI (Sigma) in PBS. Images were acquired using a confocal microscope (Leica TCS SP5 ×) with a 60 × oil objective. Images from each experiment were acquired using the same exposure time during the same imaging session. Quantification of autophagic vacuoles was analyzed by calculating the number of LC3 puncta from three fields containing more than 5 randomly selected microscopy-captured images, each comprising between 2 and 8 cells. Autophagosomes were detected as RFP+GFP+ (yellow dot), while mature, autolysosomal organelles were detected as RFP+GFP− (red-only dot) [46–48]. To examine the degree of overlap of red (Myc-Spred2) and green (GFP-LC3) spots, analysis of pixel co-localization was performed using the co-localization function of Image-Pro Plus software. The gain and offset were lowered to prevent saturation in the brightest signals. From each image three distinct areas (regions of interest, ROIs) were sampled to correct for background. Manders' overlap coefficient (R) was employed to evaluate co-localization, and therefore could be used to determine the co-localization of two signals. The values of Manders' overlap co-efficientare are in the range of 0 to 1.0. If the image has an overlap coefficient of 1, it implies that 100% of both selected channels overlap or co-localize. A value of zero means that there are no overlapping pixels. It can be used in any co-localization experiment. To assess the reliability of this method, we applied the same procedure to HeLa and COS7 cells for the co-localization of Myc-Spred2 and GFP-LC3. Quantification of the co-localization was analyzed from three fields containing more than 5 randomly selected microscopy-captured images, each of which comprised at least two cells. For each area an average of coefficients obtained from the examined field was calculated. The t-test was used to assess the results [49].

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