Question

For each primary antibody below, select what secondary antibody would have been used for fluorescent imaging in a green wavelength:

a) Mouse anti-GFAP: goat anti-mouse Alexa488 or goat anti-rabbit Alexa488 b) Rabbit anti-NF200: goat anti-mouse Alexa488 or goat anti-rabbit Alexa488 c) Rabbit anti-PSD95: goat anti-mouse Alexa488 or goat anti-rabbit Alexa488

Answer 1

a) For primary antibody Mouse anti-GFP, the secondary antibody should be the goat anti-mouse Alex 488. This is because the host for the primary antibody is the mouse. The secondary antibody's main use is in detection to achieve this it should be specific to the primary antibody.The secondary antibody is always against the host in which the primary antibody is raised. It is the Fc region of the primary antibody to which secondary antibody binds. The secondary antibody goat anti-mouse Alexa 488 is raised in goat against the mouse. Alexa-488 is a green fluorescent dye which is conjugated with secondary antibody. The dye has absorption maxima at 496 mm and emission around 519 mm and gives green color fluorescence (green wavelength 560-520 nm).

b) For primary rabbit anti-NF200, the secondary antibody should be goat anti-rabbit Alexa 488. The secondary antibody goat anti-mouse Alexa 488 can not be used as a secondary antibody it is raised in the mouse.

c) For primary antibody rabbit anti-PSD95, the secondary antibody should be the goat anti-rabbit Alexa 488. The antibody goat anti-mouse Alexa 488 can not be used as secondary because it is raised in the mouse, not in the rabbit.