Comprehensive Immunostaining Technique

Immunostaining in vivo and in vitro was performed as described previously (18 (link), 21 (link)). The samples were stained with primary and secondary antibodies as follows: anti-Rab9 mouse monoclonal antibody (1:100, Abcam, Cat #ab2810, RRID:AB_303323), anti-LAMP2 rabbit polyclonal antibody (1:100, Sigma, Cat #L0668, RRID:AB_477154), anti-LAMP2 mouse monoclonal antibody (1:100, Abcam, Cat #ab25631, RRID:AB_470709), anti-TOMM20 rabbit monoclonal antibody (1:100, Abcam, Cat #ab186734, RRID:AB_2716623), anti- LC3B rabbit polyclonal antibody (1:100, Abcam, Cat #ab51520, RRID:AB_881429), Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:100, Abcam, Cat #ab150081, RRID:AB_2734747), and Alexa Fluor 594-conjugated goat anti-mouse IgG (1:100, Abcam, Cat #ab150120, RRID:AB_2631447). Fluorescence images were obtained using an LSM700 confocal laser scanning microscope (Zeiss, Cat #LSM700, RRID:SCR_017377). Pearson's correlation coefficient was calculated to quantify the colocalization correlation of the intensity distributions between the two channels, as previously described (22 (link)). We performed independent in-vitro experiments three times.

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Uchikado Y., Ikeda Y., Sasaki Y., Iwabayashi M., Akasaki Y, & Ohishi M. (2021). Association of Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 With Angiotensin II Type 1 Receptor Impacts Mitochondrial Quality Control, Offering Promise for the Treatment of Vascular Senescence. Frontiers in Cardiovascular Medicine, 8, 788655.

Publication 2021

Alexa Fluor 594-conjugated goat anti-mouse IgG LSM700 confocal laser scanning microscope

Alexa 594 Alexa fluor 488 Anti antibody Anti igg Antibodies Confocal laser scanning microscope Fluorescence Goat Lamp2 Monoclonal antibody Mouse Rabbit Tomm20

Corresponding Organization : Kagoshima University

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Variable analysis

independent variables

In vivo and in vitro immunostaining

dependent variables

Pearson's correlation coefficient to quantify colocalization correlation of intensity distributions between two channels

control variables

Primary and secondary antibodies used for immunostaining, including anti-Rab9, anti-LAMP2, anti-TOMM20, and anti-LC3B antibodies
Alexa Fluor 488-conjugated goat anti-rabbit IgG and Alexa Fluor 594-conjugated goat anti-mouse IgG secondary antibodies
LSM700 confocal laser scanning microscope used for fluorescence imaging

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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