Immunostaining and confocal microscopy were performed as described previously [15 (link)]. Briefly, goat anti-human CRIF1 antibodies (sc-103443 and sc-103445; Santa Cruz Biotechnology, Dallas, TX, USA) were used to detect mouse CRIF1 and an anti-Tom20 antibody (#612278; BD Biosciences, San Jose, CA, USA) was used to identify the mitochondria. For immunostaining, podocytes were fixed with 4% paraformaldehyde for 15 min, permeabilized in PBS containing 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA), and then blocked with blocking buffer (10% horse serum, 1% BSA, 0.002% NaN3, and 1 × PBS) for 45 min at room temperature. Podocytes and 5 μm kidney cryostat sections were incubated with primary and secondary antibodies diluted in PBS containing 1% BSA at 4 °C overnight and room temperature for 45 min, respectively. The specimens were visualized using an LSM 510 META confocal microscope (Carl Zeiss AG, Oberkochen, Germany). All images were quantified using Image-Pro®® Plus version 6.0 software (Media Cybernetics, Silver Spring, MD, USA).
Anti tom20 antibody
Manufactured by BD
Sourced in United States
The Anti-Tom20 antibody is a protein-specific antibody that recognizes the Tom20 (translocase of outer membrane 20) protein, which is a component of the mitochondrial translocase complex. Tom20 functions as a receptor that facilitates the import of nuclear-encoded proteins into the mitochondria. The antibody can be used to detect and study the localization and expression of Tom20 in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Triton x 100, by Merck Group (1 mentions)
Image pro plus version 6, by Media Cybernetics (1 mentions)
Alexa fluor 488 anti rabbit, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342 ho, by Merck Group (1 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
Dihydroethidium he, by Thermo Fisher Scientific (1 mentions)
Anti lc3b antibody, by Merck Group (1 mentions)
Anti mouse alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
2 protocols using anti tom20 antibody
1
Immunostaining and Confocal Microscopy Protocol
2
Mitochondrial and Autophagic Dynamics Imaging
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Cells were seeded on coverslips pretreated with poly-Llysine (Sigma-Aldrich, P4707), fixed with paraformaldehyde (PFA; 4% w:v; Fluka-Chemical, 76240), permeabilized with Triton X-100 solution (0.1% v:v in phosphate-buffered saline 1X; Sigma-Aldrich, T9284), and stained with an anti-Tom20 antibody (BD Biosciences, 556432) to measure mitochondrial morphology or an anti-LC3B antibody (Sigma-Aldrich, L7543) to visualize autophagic structures. The cells were then incubated with Alexa Fluor 568 antimouse (Invitrogen, A11031) or Alexa Fluor 488 anti-rabbit (Invitrogen, A11008) secondary antibodies. Chromatin condensation was labeled with Hoechst 33342 (Ho, 2 µM; Sigma-Aldrich, 101174857). For reactive oxygen species (ROS) quantification, cells were stained in vivo with dihydroethidium (He, 5 µM; Invitrogen, D1168) for 15 min at 37 °C, and they were then fixed with PFA. To measure lysosome size, cells were labeled in vivo with Lysotracker Red DND-99 (LTR, 2 µM; Invitrogen, L7528) for 15 min at 37 °C and then fixed with PFA. Fluorescence images were analyzed with an inverted fluorescence microscope (IX51, Olympus) equipped with a camera (DP70, Olympus).
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