Mouse serum negative control - New to immunohistochem. Need some help/advise (Jun/22/2010 )
Hi! I've just started doing some immunohistochemistry on paraffin embedded human lung sections and need some help/advise on choosing a suitable negative control.
The primary antibody that i use is a mouse polyclonal raised against a human protein (MS4A8B protein). I have used mouse serum as my negative control and perform a similar dilution as my primary Ab (1 in 50).
Strangely, when i got my results, i noticed that there is heavy staining virtually everywhere on my section with the mouse serum when its actually supposed to show no staining! I am puzzled why this has occurred. Could it be that i am using an innapropriate dilution? Also, i don't know if this would affect my results but i have accidentally left the mouse serum in the fridge (4 celcius) for a few days instead of keeping it frozen in the freezer.
Any help/advise would be most useful. Thank you.
The mouse ab that you use maybe purified and specific for the antigen in your sample. (Are you sure it is polyclonal...rather than Mab?).
the mouse serum you use as negative may contain abs and other proteins that react with human antigens and other proteins present in your sample. the serum is also not exactly a negative control. I negative control would be non-specific mouse antibody of the same purity as your primary and non cross reactive with human proteins.
Thus, when you run your test the same concentration of antibodies in the same matrix are added.
Hi. I'm sure the mouse antibody that i've used is a polyclonal antibody.
Also, the reason why i've used mouse serum as my negative control is because i'm following a method performed from a staff previously. From her results, mouse serum showed no staining.
With the mouse serum issue aside, what would you recommend i use as a suitable negative control? Specifically the sections i'm using are human bronchi lung sections embedded in paraffin and my antibody stains most strongly on the epithelial surface and a little bit on the smooth muscle.
Thanks for your input/advise!
obtain some purified mouse IgG. Expressing protein concentrations by dilution is incorrect (ie "1:50" etc). With both your primary ab and your purified control determine OD 280 to obtain concentrations in mg/ml and use both of them at the same concentration (ie you will convert your '1:50' dilution to mg/ml and your control will be used at this same concentration.)
Your conjugate should be specific for mouse IgG and not react with human proteins. Be sure to test the dilution you use on a slide without the mouse abs to see if there is any binding and titer accordingly.