Rabbit anti calreticulin

Whole-cell lysates and cell fraction lysates were prepared as described above, and protein concentrations were determined by the Bradford method (Bradford, 1976 (link)). Samples were diluted in a reducing sample buffer, and subjected to SDS-PAGE using Peirce pre-cast Tris-HEPES gels. A BioRad SemiDry Transfer apparatus was used for gel transfer to PVDF. The resulting Western blots were probed using the following primary antibodies: PRK8 mouse-anti-Parkin (1:200; Santa Cruz), rabbit-anti-Mitofusin-2 (1:2000; Sigma, cat# 110M4842), mouse-anti-HSP60 (1:1200; Stressgen, cat# SPA-806), rabbit-anti-calreticulin (1:1000; Abcam, cat# ab92516), rabbit-anti-GAPDH (1:1200; Santa Cruz, cat# sc25778), rabbit-anti-actin (1:12,000; Abcam, cat# ab8227), rabbit-anti-FACL4 (1:5000; Abcam, cat# ab137525). For secondaries, corresponding Li-Cor Odyssey compatible IR680- and IR800-conjugated antibodies were used for detection, and blots were imaged and analyzed using a Li-Cor Odyssey system (Li-Cor).

Floating sections were incubated in blocking solution (PBS, 0.05% bovine serum albumin, 2% fetal bovine serum, 1% Triton X-100 and 0.1% saponin) for 30 min prior to addition of primary antibodies: chicken anti-GFP (1:1000, Abcam, Cambridge, MA, USA), mouse anti-FLAG (1:500, Millipore Sigma, St. Louis, MO, USA), rabbit anti-CHCHD3 (1:100, Proteintech, Rosemont, IL, USA), rabbit anti-calreticulin (1:250, Abcam, Cambridge, MA, USA), chicken anti-Map2 (1:200, Abcam, Cambridge, MA, USA), and rabbit anti-transportin 1 (1:250, Abcam, Cambridge, MA, USA) at 4 °C overnight. After PBS washes, sections were incubated in appropriate secondary antibodies in blocking solution: Alexa Fluor 488 (1:1000, Thermo Fisher Scientific, Rockford, IL, USA), Alexa Fluor 647 (1:1000, Thermo Fisher Scientific, Rockford, IL, USA) and Cy3-conjugated (1:1000, Molecular Probes, Eugene, OR, USA) for 2 h at room temperature (RT). Antigen retrieval with 0.01 M sodium citrate, pH 9, for 3 h in a water bath at 80 °C was performed prior to blocking for rabbit anti-RanGap1 antibody (1:250; Abcam, Cambridge, MA, USA) as previously described [14 (link)]. Sections were counterstained with DAPI (1:5000). All sections were mounted on slides and coverslipped with Fluoromount G (Electron Microscopy Sciences, Hatfield, PA, USA).