[Histonet] Re: lab math: ihc dilution

[Teri Johnson]

Here's how I approach this problem.

Convert all concentrations into the same units. I prefer to use whichever the final concentration unit is, so it looks like this:
1.56 mg/ml = 1560 ug/ml

You have 1560 ug/ml and you need 0.8 ug/ml
Divide 1560 by 0.8 = 1950. So your final dilution is 1:1950.

Pipetting 1 ul of antibody in a dilution isn't very accurate, and using the above  as 1 ul antibody in 1949 ul of diluent would yield you almost 2 ml of diluted antibody. Not a problem if  you are doing batch staining where you might need 5 or 10 ml of diluted antibody. But if you are staining only 1 or 2 slides, you might not want to make that much.

So follow Liz's advice about starting with a 1:10 dilution. Short division (move the decimal point one place to the left) puts the concentration at 156.0 ug/ml.

156 divided by 0.8 - 195 (You can see from above you could have just moved the final dilution decimal point one place to the left to get the same answer)

With a 1:10 dilution of stock your final dilution would be 1:195 and that seems a bit more reasonable for fewer slides/total volume. Remember that 1:195 dilution is 1 part of antibody added to 194 parts of diluent.

I would then multiply each side by the same number to get roughly how ever much diluted antibody you need. You can make *EXACTLY* however much you need, but that requires more complicated math, and I don't mind making just slightly over what I need to account for pipetting loss.

Ex: If I need 100 ul/slide of diluted antibody and 4 slides to stain, I would multiply both sides by 3, since multiplying it by 2 won't give you quite enough :
1 x 3 = 3 (ul of 1:10 antibody)
194 x 3 = 582 (ul of diluent)

Not sure if I helped or confused things.

Teri Johnson, HT(ASCP)QIHC
Manager Clinical Trial Testing
Genoptix, Inc.
SAN5, Rm. 2005
760.516.5954 (office)
760.516.6201 (fax)


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