Anti mouse igg alexa fluor 488 for green

Cardiac paraffin sections were deparaffinized as mentioned above. Then, the co-immuno localization of 8OHG and OGG-1 with mitochondrial markers TOM20 and aconitase respectively was performed. First, the double immunostaining with primary antibodies of 8OHG; anti-mouse 8OHG antibody (1:100) (Abcam) and Tom 20; anti-rabbit TOM 20 antibody (SantaCruz) (1:100) were used along with respective secondary antibodies, anti-mouse IgG Alexa Fluor 488 for green and anti-Rabbit IgG Alexa Fluor 568 for red (Thermo Fischer). Similarly, for the double staining of OGG-1 and aconitase, the primary antibodies: anti-rabbit OGG-1 antibody (Novus Biologicals, LLC Centennial CO) and anti-mouse aconitase antibody (Abcam) along with respective secondary antibodies, anti-rabbit IgG Alexa Fluor 488 for green and anti-mouse IgG Alexa Fluor 568 for red (Thermo Fischer) were used. See above for microscopic and imaging details.

Following standard deparaffinization protocol, co-localization of 4HNE with OGG1 and 4HNE with 8OHG was performed by using double immunostaining with 4-HNE-Cys/His/Lys (Millipore Sigma) rabbit antibody (1:200) and OGG-1 mouse monoclonal IgG antibody (1:100) (Santa Cruz Biotechnology, Santacruz, CA). Then, we used secondary antibodies anti-Rabbit IgG Alexa Fluor 568 for red and anti-mouse IgG Alexa Fluor 488 for green (Thermo Fisher Scientific) respectively. Next, for the co-localization of 4HNE with 8OHG, 4-HNE-Cys/His/Lys rabbit antibody (Millipore Sigma) (1: 100) and anti-mouse 8OHG antibody (1: 100) (Abcam) with corresponding secondary antibodies; goat anti-Rabbit IgG Alexa Fluor 488 for green and donkey anti-mouse IgG Alexa Fluor 568 for red were used. Histologic micrograph images were captured using Olympus IX81 (Olympus America, Center Valley, PA). The images were captured with a digital camera (DP72) and DPC controller software (Olympus America, Center Valley, PA) set at 40x magnification. The details of fluorescent single band filters are as follows:
Blue (DAPI) excitation wavelength (Ex): 360–370nm & emission wavelength (Em): 420–460nm; Green (Alexa Flour 488) Ex:460–500nm & Em: 510–560nm; Red (Alexa Flour 488) Ex:533–587nm & Em: 607–682nm. These are same for all florescence imaging done.