I have a primary antibody with size: 50 μg and concentration 0,5mg/ml and I shoud have used to IHC - Paraffin (3.75 µg/ml). I understand that in this case I should use a ratio of 1: 133?
Obviously, this is the suggested value from the manufacturer, in fact this ratio may be different. I just wanted to ask if my way of thinking is correct.
Best wishes
Hello Izabela
If you feel the need to change the value suggested by the manufacturer then you should use a good positive control for selecting the proper primary antibody concentration for IHC.
Usually for IHC you can check 1:100, 1:200 and 1:400 dilutions for the primary antibody. Whichever dilution gives a clear staining pattern with minimal background staining can be used.
All the best.
Malcom is correct. Here is the math, your stock is 500 ug/ml and you need to dilute it to 3.75 ug/ ml, so that is really 500/3.75 or 1:133. Using a range as suggested by Malcom will allow you to optimize using your reagents, sample, method. Ihc can be a little tricky if you are using DAb, if you wait too long to stop the chromate reaction, everything will look brown, so be prepared to factor in a learning curve.
Malcolm Nobre and Lora Benoit thank you so much for your answers.
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