Question (39707) | Goat Anti-Mouse IgG H&L (DyLight® 488) (ab96871)

Go to datasheet (ab96871)

Question

This customer has purchased ab96885 (Goat anti-Rabbit IgG H&L (DyLight® 594) secondary antibody), ab96871 (Goat anti-Mouse IgG H&L (DyLight® 488) secondary antibody) and has conducted the ICC several times. This customer indicated she will observe the PE fluorescence when she wants to observe the FITC fluorescence, but this state doesn’t happen when she wants to observe the PE fluorescence, therefore, she would like to ask for your help to find out any possible problem and also help her to modify her experiment step, could you please offer any suggestion to her?

I attached the image in this letter and her experiment step as follow:





1) Order details:

˙Batch number (Lot number): ab96885--gr46891-6 and ab96871--gr50260-4

Po: 1028918

˙Abcam product code: ab96885, ab96871

˙Antibody storage conditions (temperature/reconstitution etc) : 4℃



2) Please describe the problem (high background, non-specific signal…etc).

I can observe the PE fluorescence when I want to observe the FITC fluorescence

3) On what material are you testing the antibody in IHC/ICC?

˙Species: 1% BSA, in 1X PBS

˙What’s cell line or tissue: sclera cell



4) Sample preparation:

˙4%PFA/formalin fixed

˙cells in culture

˙Sample preparation:

˙Positive control :

˙Negative control :



5) Fixation step

˙Yes or No : Yes

˙If yes, Fixative agent and concentration: 4%PFA/formalin fixed

˙Fixation time: 5min

˙Fixation temperature :R.T.



6) Antigen retrieval method(including time, temperature etc.):

R.T. 60min



7) Permeabilization method:

˙Did you do a permeabilization step (details please) or add permeabilizing agent in any dilution buffers?

˙Permeabilizing agent and concentration:





8) Blocking agent (eg BSA, serum…):

˙Concentration:1% BSA

˙Blocking time : 60min

˙Blocking temperature: R.T.



9)

˙Endogenous peroxidases blocked?

˙Endogenous biotins blocked?



10) Primary antibody (If more than one was used, describe in “additional notes”) :

˙Species:

˙Reacts against:

˙At what dilution(s) have you tested this antibody:1:50˜250

˙Diluent buffer: 1% BSA in 1xPBS

˙Incubation time: 60min

˙Incubation temperature: R.T.

˙What washing steps were done (which buffer, number of washes): 3times, 5min



11) Secondary antibody:

˙Species:

˙Reacts against which species:

˙At what dilution(s) have you tested this antibody: 1:1000

˙Diluent buffer: 1% BSA in 1xPBS

˙Incubation time: 60min

˙What washing steps were done (which buffer, number of washes): 3times, 5min

˙Fluorochrome or enzyme conjugate (eg: FITC, HRP, AP, biotin…etc): FITC and TRITC

˙Do you know whether the problems you are experiencing come from the secondary?



12) Signal amplification method (eg: ABC, LSAB, HRP polymer, TSE):



13) Detection method (eg: DAB, BCIP/NBT …etc):



14)

˙ How many times have you run this staining? 2

˙Do you obtain the same results every time? YES

˙What steps have you altered to try and optimize the use of this antibody?





Could you please help this customer to solve the problem?

Thanks for your kindly help

Answer

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimize the results from abab96885 Goat anti-Rabbit IgG H&L (DyLight® 594) secondary antibody. I would also appreciate if you can confirm some further details:

1. What kind of filter or lamp are you using? Unfortunately, DyLight® 488and DyLight® 594 do overlap: a little bit in the DyLight® 488 spectrum, but more important in the DyLight® 594 wavelengths, which corresponce with your findings. Check with the manufacturer of your microscope if their filters are suitable for those dyes.

2. In order to check if the ab96885 is damaged I would suggest to perform an ICC experimentwith only one primary- secondaryreaction. This way you canexclude the annoying "bleed-over".


Should the suggestions not improve the results, please do let me know.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

Sign up