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Primary antibody dilution in IHC (immunohistochemistry) studies
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+ Western blot
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Posted by
Aaron Turpen
Primary antibody dilution in IHC (immunohistochemistry) studies
Thank you everyone for your really detailed and helpful replies,
I've read your answers carefully. They are very appreciated. Bottom line is that it's crucial to provide an optimal signal with low background. I can understand the fact that because volumes differ between two methods, the amount of antibody compounds of each volume would be more similar. I've also learned that I don't have to be stuck at the suggested ratio. It might be a better idea to test different dilutions for the best dilution ratio. Ivana Vrhovac Madunic The suggestions on optimal staining are very important to me. I've just finished reading these three papers you provided. I didn't know that an antibody amplification system can allow a higher dilution of primary antibody. They are all quite helpful. Thank you very much again.
Thank you everyone for your really detailed and helpful replies,
I've read your answers carefully. They are very appreciated. Bottom line is that it's crucial to provide an optimal signal with low background. I can understand the fact that because volumes differ between two methods, the amount of antibody compounds of each volume would be more similar. I've also learned that I don't have to be stuck at the suggested ratio. It might be a better idea to test different dilutions for the best dilution ratio. Ivana Vrhovac Madunic The suggestions on optimal staining are very important to me. I've just finished reading these three papers you provided. I didn't know that an antibody amplification system can allow a higher dilution of primary antibody. They are all quite helpful. Thank you very much again.
Yes, besides the factors mentioned in the previous answers, it is always recommended to test a new antibody (or tissue from a new experimental setting) using series of dilutions. The best dilution is sometimes quite far from those recommended by the manufacturer. (In our studies we are usually using the dilutions of the primary ab ranging from 1:100 til 1:20000, depending on used Ab or type of the tissue).
Yes, besides the factors mentioned in the previous answers, it is always recommended to test a new antibody (or tissue from a new experimental setting) using series of dilutions. The best dilution is sometimes quite far from those recommended by the manufacturer. (In our studies we are usually using the dilutions of the primary ab ranging from 1:100 til 1:20000, depending on used Ab or type of the tissue).
With Western blotting, one usually has a large tank of antibody compared to the volumes (a droplet or two) which are used for immunocytochemistry. If you counted up the number of antibody molecules in the volume, they are not exactly the same but would be more similar than suggested by the ~10-fold difference in dilution factor.
With Western blotting, one usually has a large tank of antibody compared to the volumes (a droplet or two) which are used for immunocytochemistry. If you counted up the number of antibody molecules in the volume, they are not exactly the same but would be more similar than suggested by the ~10-fold difference in dilution factor.
Hi Gokhan, You are right! There is no need to use manufacture's dilution for antibodies. You can use whichever dilution work for your tissue or cells. Also, manufactures often suggest to determine concentration experimentally. -Kazi
Hi Gokhan, You are right! There is no need to use manufacture's dilution for antibodies. You can use whichever dilution work for your tissue or cells. Also, manufactures often suggest to determine concentration experimentally. -Kazi
Well, the dilution factors of 1:100; 1:200 for IHC and 1:1000 and 1:2000 usually coming from commercial products frequently sold with 0.5-2 mg/ml (5-20 ug/ml) for IHC and 0.5-2 ug/ml for WB. However, with the antibody, the efficacy also comes from the affinity of how well antibodies bind with the target thus antibody with poor affinity would not produce a strong signal even with 1:10 concentration, and antibodies with high affinity may need higher dilution to avoid overexposure or burning the membrane. Overall, 1:100 is a reasonable starting dilution point for most commercial antibodies with a final concentration that should be adjusted based on the experimental observations.
Well, the dilution factors of 1:100; 1:200 for IHC and 1:1000 and 1:2000 usually coming from commercial products frequently sold with 0.5-2 mg/ml (5-20 ug/ml) for IHC and 0.5-2 ug/ml for WB. However, with the antibody, the efficacy also comes from the affinity of how well antibodies bind with the target thus antibody with poor affinity would not produce a strong signal even with 1:10 concentration, and antibodies with high affinity may need higher dilution to avoid overexposure or burning the membrane. Overall, 1:100 is a reasonable starting dilution point for most commercial antibodies with a final concentration that should be adjusted based on the experimental observations.
For each usage of the primary antibody optimization of concentration is necessary. From experience in my lab, even if you use it in different organs/tissues even animals/human samples, you have to find the optimal concentration of the primary antibody to determine optimal signal with low background noise. This also refers to secondary antibody (they are usually with higher titer so you dilute it more). Furthermore, for optimal staining you have to pay attention to antigen retrieval protocols (incubation time, temperature, pH etc.) and as well to have positive and negative control to be sure your primary antibody is specific. I wish you good luck!
For each usage of the primary antibody optimization of concentration is necessary. From experience in my lab, even if you use it in different organs/tissues even animals/human samples, you have to find the optimal concentration of the primary antibody to determine optimal signal with low background noise. This also refers to secondary antibody (they are usually with higher titer so you dilute it more). Furthermore, for optimal staining you have to pay attention to antigen retrieval protocols (incubation time, temperature, pH etc.) and as well to have positive and negative control to be sure your primary antibody is specific. I wish you good luck!
Thank you everyone for your really detailed and helpful replies,
I've read your answers carefully. They are very appreciated. Bottom line is that it's crucial to provide an optimal signal with low background. I can understand the fact that because volumes differ between two methods, the amount of antibody compounds of each volume would be more similar. I've also learned that I don't have to be stuck at the suggested ratio. It might be a better idea to test different dilutions for the best dilution ratio. Ivana Vrhovac Madunic The suggestions on optimal staining are very important to me. I've just finished reading these three papers you provided. I didn't know that an antibody amplification system can allow a higher dilution of primary antibody. They are all quite helpful. Thank you very much again.
Kind regards,
Thank you everyone for your really detailed and helpful replies,
I've read your answers carefully. They are very appreciated. Bottom line is that it's crucial to provide an optimal signal with low background. I can understand the fact that because volumes differ between two methods, the amount of antibody compounds of each volume would be more similar. I've also learned that I don't have to be stuck at the suggested ratio. It might be a better idea to test different dilutions for the best dilution ratio. Ivana Vrhovac Madunic The suggestions on optimal staining are very important to me. I've just finished reading these three papers you provided. I didn't know that an antibody amplification system can allow a higher dilution of primary antibody. They are all quite helpful. Thank you very much again.
Kind regards,
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Yes, besides the factors mentioned in the previous answers, it is always recommended to test a new antibody (or tissue from a new experimental setting) using series of dilutions. The best dilution is sometimes quite far from those recommended by the manufacturer. (In our studies we are usually using the dilutions of the primary ab ranging from 1:100 til 1:20000, depending on used Ab or type of the tissue).
Yes, besides the factors mentioned in the previous answers, it is always recommended to test a new antibody (or tissue from a new experimental setting) using series of dilutions. The best dilution is sometimes quite far from those recommended by the manufacturer. (In our studies we are usually using the dilutions of the primary ab ranging from 1:100 til 1:20000, depending on used Ab or type of the tissue).
More
VOTE
With Western blotting, one usually has a large tank of antibody compared to the volumes (a droplet or two) which are used for immunocytochemistry. If you counted up the number of antibody molecules in the volume, they are not exactly the same but would be more similar than suggested by the ~10-fold difference in dilution factor.
With Western blotting, one usually has a large tank of antibody compared to the volumes (a droplet or two) which are used for immunocytochemistry. If you counted up the number of antibody molecules in the volume, they are not exactly the same but would be more similar than suggested by the ~10-fold difference in dilution factor.
More
VOTE
Hi Gokhan,
You are right! There is no need to use manufacture's dilution for antibodies. You can use whichever dilution work for your tissue or cells. Also, manufactures often suggest to determine concentration experimentally.
-Kazi
Hi Gokhan,
You are right! There is no need to use manufacture's dilution for antibodies. You can use whichever dilution work for your tissue or cells. Also, manufactures often suggest to determine concentration experimentally.
-Kazi
More
VOTE
Well, the dilution factors of 1:100; 1:200 for IHC and 1:1000 and 1:2000 usually coming from commercial products frequently sold with 0.5-2 mg/ml (5-20 ug/ml) for IHC and 0.5-2 ug/ml for WB. However, with the antibody, the efficacy also comes from the affinity of how well antibodies bind with the target thus antibody with poor affinity would not produce a strong signal even with 1:10 concentration, and antibodies with high affinity may need higher dilution to avoid overexposure or burning the membrane. Overall, 1:100 is a reasonable starting dilution point for most commercial antibodies with a final concentration that should be adjusted based on the experimental observations.
Well, the dilution factors of 1:100; 1:200 for IHC and 1:1000 and 1:2000 usually coming from commercial products frequently sold with 0.5-2 mg/ml (5-20 ug/ml) for IHC and 0.5-2 ug/ml for WB. However, with the antibody, the efficacy also comes from the affinity of how well antibodies bind with the target thus antibody with poor affinity would not produce a strong signal even with 1:10 concentration, and antibodies with high affinity may need higher dilution to avoid overexposure or burning the membrane. Overall, 1:100 is a reasonable starting dilution point for most commercial antibodies with a final concentration that should be adjusted based on the experimental observations.
More
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yes, i am with this opinion thank you
yes, i am with this opinion thank you
More
VOTE
For each usage of the primary antibody optimization of concentration is necessary. From experience in my lab, even if you use it in different organs/tissues even animals/human samples, you have to find the optimal concentration of the primary antibody to determine optimal signal with low background noise. This also refers to secondary antibody (they are usually with higher titer so you dilute it more). Furthermore, for optimal staining you have to pay attention to antigen retrieval protocols (incubation time, temperature, pH etc.) and as well to have positive and negative control to be sure your primary antibody is specific. I wish you good luck!
Literature that may help: Chapter Role of microwave heating in antigen retrieval in cryosectio...
Article Expression profiling and immunolocalization of Na(+)-D-gluco...
Article Proper Application of Antibodies for Immunohistochemical Det...
For each usage of the primary antibody optimization of concentration is necessary. From experience in my lab, even if you use it in different organs/tissues even animals/human samples, you have to find the optimal concentration of the primary antibody to determine optimal signal with low background noise. This also refers to secondary antibody (they are usually with higher titer so you dilute it more). Furthermore, for optimal staining you have to pay attention to antigen retrieval protocols (incubation time, temperature, pH etc.) and as well to have positive and negative control to be sure your primary antibody is specific. I wish you good luck!
Literature that may help: Chapter Role of microwave heating in antigen retrieval in cryosectio...
Article Expression profiling and immunolocalization of Na(+)-D-gluco...
Article Proper Application of Antibodies for Immunohistochemical Det...
More
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