[Histonet] Re: PLS Help with IHC protocol

[Johnson, Teri]

Kimberly, you asked:

>>Step 10: Add antibody at 10ug/ml in PBS + 0.1%BSA + 1:10 volume CAS block

Can someone break this down for me in simple instructions?<<
And
>>Its 1.1mg/ml

Another question: they are asking that I add glycerol to the antibody to prevent freezing. Wont that change my concentration?<<

Some antibody companies request you add equal parts of glycerol to the antibody stock to prevent freeze/thaw cycles which is not good for antibody integrity. If you add equal parts of glycerol, your concentration would now be .55 mg/ml.  In order to get ug/ml, you will need to convert .55 mg/ml to ug/ml. You do that by multiplying by 1000, therefore .55 mg/ml = 550 ug/ml. In order to get 10 ug/ml concentration, you would divide 550 by 10, and you get 55. So you would use a 1:55 dilution of antibody/glycerol stock. The PBS/BSA/CAS block is a stock solution which you will then use as the diluent for the antibody. Some folks make it up using commercial antibody diluent. Some folks use whatever they use for the normal serum block (say 5-10% serum from the species source the secondary antibody is made in, i.e. normal goat serum if your secondary antibody is goat anti-rabbit). There is no one best diluent for antibodies. If you want to use the PBS/BSA/CAS block, go ahead. But you don't have to if you want to simplify things.

Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110