Dilutions and immunohistochemistry

[EveC]

Hello,

I would have a question about dilutions in immuohistochemistry. Maybe could help me?

The desired dilution is 1: 100. The first protocol that I used said to add 10ul in 1000ul buffer. I considered this too much antibody, but, If I am right, it is still correct since 1000/10=100 dilution factor.

After that, a new protocol was available, and this last protocol said to do 1:100 dilution and prepare 500 ul of primary antibody solution. So I prepared  5ul antibody and 500 ul  buffer (since 500/5 = 100, dilution factor).

However, I see that some people consider that it has to be 5ul and 495ul of buffer to reach the final volume of 500 (or in the first case 10ul antibody and 990ul buffer).

Which of these ways to do is valid? Is this correct to add 5ul of Antibody in 500ul of buffer to have a dilution of 1:100?  I am confused concerning total volume.

Thank you very much for your time and help.

I look forward to hearing from you.

[studiot]

1 hour ago, EveC said:

After that, a new protocol was available, and this last protocol said to do 1:100 dilution and prepare 500 ul of primary antibody solution. So I prepared  5ul antibody and 500 ul  buffer (since 500/5 = 100, dilution factor).

This is not correct as it is 5 in 505 which is not the required ratio 1 : 100.

1 hour ago, EveC said:

However, I see that some people consider that it has to be 5ul and 495ul of buffer to reach the final volume of 500 (or in the first case 10ul antibody and 990ul buffer).

This is correct as it is the required ration of 5 in 500 or 1:100.

There are several ways to work this out:

Here are same example calculations working out the same example by different methods.

 

 

[CharonY]

Studiot is correct. I will say that depending on application and especially if the total volume does not change much and/or the pipetting might be large relative to the error in the dilution (e.g. if you pipette 1 ul into 1000 ul instead of 1 ul in 999ul)  some folks just round up.

That, however, is a bit sloppy and if folks do it without proper consideration, it can affect the results (especially for quantitative analyses). 

Moreover in many student protocols (and annoyingly some papers) I have seen confuse the dilution factor with dilution ratio. I can only recommend that if you write a thesis that you clarify exactly what you mean in your materials and methods section.

[EveC]

Hello everyone, and thank you very much for your very helpful responses. The confusion came mainly from the way some people say: "a dilution of 1: 100  so add 3ul in 300ul of buffer", which is not accurate, as you mention.

However, in some biological techniques, the precision also depends on the method and objective, as you also precise. In particular in histology (I am working for now with histology techniques). This is the case in immunohistochemistry.

For an immunofluorescence assay, can this type of rounding be done?

Thank you very much and have a nice day.

[studiot]

43 minutes ago, EveC said:

Hello everyone, and thank you very much for your very helpful responses. The confusion came mainly from the way some people say: "a dilution of 1: 100  so add 3ul in 300ul of buffer", which is not accurate, as you mention.

However, in some biological techniques, the precision also depends on the method and objective, as you also precise. In particular in histology (I am working for now with histology techniques). This is the case in immunohistochemistry.

For an immunofluorescence assay, can this type of rounding be done?

Thank you very much and have a nice day.

I would listen to Charony's comment on this when it comes as hsitology is not my subject.

I would just say that in Science generally it is always right to say what you mean exactly. Experts in a particular area may take short cuts but they may also then be misunderstood.

Here is a humerous sketch that really shows this.

 

[EveC]

Hello, thanks for your reply. Yes, of course, you have to be precise in science and agree with a misunderstanding when asking people.

Other techniques, for example, in molecular biology, will need all the precision, and this type of rounding affects the result.

The immunofluorescence worked to show the presence of a protein in the tissue region. For now, I have not done a quantification with the IF. However, in the future, it is better to be careful with this rounded.

Thank you and have a nice weekend!