immunochemical staining problem - i'm struggling to resolve this problem (Aug/26/2010 )

Hi

I want to see per1(flag tag) protein localization after co-expressing CSNK1A (Ha tag).
Primary antibody are Anti-HA in rabbit, Monoclonal ANTI-FLAG, secondary antibody are Anti-mouse IgG 488, 2mg/ml, Anti-rabbit IgG 568, 2mg/ml. green channel is working well but red channel is not working(nothing stained, but DAPI shows nuclei is good).
Then I change the primary and secondary antibody:
Primary antibody are Anti-HA in mouse, Anti-Flag in rabbit, secondary antibody are Anti-mouse IgG 568, 2mg/ml, Anti-rabbit IgG 488, 2mg/ml. red channel is still not working(nothing stained but DAPI shows nuclei is good).
Thank you so much for you suggestion!
Best,
Leo


Primary antibody dilution Second antibody dilution
Anti-HA in rabbit From 1:200 Anti-rabbit IgG 568, 2mg/ml From 1:200
Anti-Flag in rabbit From 1:200 Anti-rabbit IgG 488, 2mg/ml From 1:200
Anti-HA in mouse From 1:200 Anti-mouse IgG 488, 2mg/ml From 1:200
Monoclonal ANTI-FLAG From 1:200 Anti-mouse IgG 568, 2mg/ml From 1:200

Your antibody concentrations are very high, try diluting them to 1:2000ish, it should be a lot more specific. Red fluorescence is often much "fainter" than green - so you may not be able to see it with the naked eye. Try taking a long exposure picture using a camera attached to the microscope. Are you sure that the correct bandpass filter is being used for exciting the fluorophore?