Rabbit anti beclin1

To visualize Bm16M intracellular trafficking, the indicated host cells (5.0 × 10⁴) were seeded on 12-mm coverslips placed on the bottom of wells of 24-well plates and infected with Bm16M-GFP or Bm16M for various lengths of time. To analyze infections of less than 0.5 h, the infected cells were rinsed with 1 × PBS and then fixed with 3.7% formaldehyde at 4°C for overnight before immunofluorescence microscopy analysis (IFMA). Otherwise, at 0.5 h.p.i., the infected cells were washed with 1 × PBS and fresh media supplemented with 40 μg/ml gentamicin was added to kill extracellular bacteria. At the indicated time points post-infection, the infected cells were fixed and IFMA was performed as previously described (Qin et al., 2008 (link), 2011 (link); Pandey et al., 2017 (link)). The primary antibodies used were as follows: goat-anti Brucella, rabbit anti-LAMP-1; rabbit anti-cathepsin D; rabbit anti-mouse LC3; rabbit anti-Calreticulin; rabbit anti-ULK1, rabbit anti-Beclin 1 (Santa Cruz Biotech., Inc, 1:200-500). Samples were stained with Alexa Fluor 488-conjugated and/or Alexa Fluor 594-conjugated secondary antibody (Invitrogen/Molecular Probes, 1:1,000). Acquisition of confocal images, and image processing and analyses were performed as previously described (Qin et al., 2008 (link), 2011 (link); Pandey et al., 2017 (link)).

NSC34 cells were seeded on coverslips and grown overnight. Briefly, cells were transfected and after 24 h cells washed in PBS, fixed with 4% paraformaldehyde (Merck) for 10 min at room temperature, permeabilized with 0.1% Triton X-100 (Merck, 9036-19-5) and 5% gelatin from cold water fish (Sigma, 9000-70-8) for 2 h at room temperature in a moisture chamber. Antibodies and concentrations employed were: mouse Pacer (custom-made from Abmart), 1:100; rabbit anti-Beclin1 (Santa Cruz Biotechnology, sc-11,427), 1:100. Secondary antibodies were used as follows: anti-mouse Alexa 555 (Thermo Fisher Scientific, A28180), 1:1000 and anti-rabbit Alexa 488 (Thermo Fisher Scientific, A27034), 1:1000. Nuclei were stained with Hoechst 33342 (Life Technologies, H3570)1:1000. Coverslips were mounted with Fluoromount G. Fixed cells were imaged with a Leica TCS SP8 confocal microscope. ImageJ and LAS X were employed to process the stacked images. The co-localization analysis was performed as in [42 (link)]. Briefly, confocal images were processed using CDA software. Applying the same thresholds for all experimental conditions, the software provides a coefficient of colocalization (Pearson’s correlation coefficient), which was used comparatively.