LacZ background - LacZ background on frozen sections (May/11/2004 )

Hi, i've just lacZ stained some frozen sections of adult mouse tissues (kidney, spleen and lung) and i've got lacZ background on both my transgenic and wildtype tissues as well as (it looks like) specific staining on my transgenic samples. I was wondering if anybody has a protocol or suggestion for eliminating lacZ background. Thanks!

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Hi, if you are trying to detect the activity of the reporter gene LacZ and have a lot of background, may be the problem it's that your are detecting the endogenous b-galactosidase of your sample (and the samples you say are dealing with, seem to me to have it). There is one easy solution for that, changing the pH of your buffer above 8.5. The b-galactosidase activity from most animals has its optim pH around 4-5, .... but they are not active over more alkaline conditions, whereas the bacterial enzime still is active. In this way, although you'll decrease the efficiency of your lacZ detection, you will get rid of all background. There is this refference just in case it's useful for you. Dev Genes Evol (2001) 211:154–156.
Hope it works.
Good luck

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