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IF staining questions
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IF staining questions


Why would one decide to use a primary antibody along with a secondary antibody rather than a conjugated primary antibody?

Is it better to buy and use a primary antibody conjugated with Alexafluor488 or to do the primary and a Alexafluor488 conjugated secondary antibody?

Answer 1.

If you want a stronger signal I would probably use the secondary antibody vs using a directly conjugated primary antibody. You could use a directly conjugated one as you would for flow cytometry but it won't give as much amplification as it would with a secondary step built in.

Katy Milne

Answer 2.

Direct IF (a fluorescent conjugated primary) is certainly easier, but there are some reasons one might prefer using an indirect method (using a conjugated secondary). Indirect methods allow the use of a different wavelength to be used simply by switching the secondary. It is also possible for the fluorescent tag to fade or not be strong enough to start with. The secondary could be used at a higher concentration or replaced in the case of a faded primary.

Amos Brooks

Answer 3.

I'd like to preface that I haven't done any Alexaflour testing in just over 10 years. The simplest response may prefer using a conjugated secondary if you're planning to do multiplex or multicolor analysis. If I was limited in commercially available options for primaries needed for the test sample, then i would prefer a conjugated secondary as it loosens up testing requirements for testing. Another benefit is that it may increase specific testing and allow positive staining to appear more visibly present than a directly conjugated antibody. There are other benefits for a conjugated secondary or tertiary antibody, but it requires users to know how to troubleshoot the stain. Direct conjugation is easier to use in many circumstances. I would communicate to your friend to try both for the study and determine as a team of internal collaborators on which method is easiest and adequate enough for performing the method testing. I hope this helps.

Eddie Martin

Answer 4.

The primary antibody is the most expensive reagent. If you use the primary ab unconjugated and visualize using a conjugated secondary you will significantly increase the dilution factor of the primary ab thus your primary ab will last much longer (saving you money and maybe less background noise). Same principle applies to using antigen retrieval ( AR) vs no ag AR: eg: I can use a particular ab to detect "endogenous" GFP (actually tagged to a protein) in paraffin sections without using AR ( actually HIER) at a dilution factor of 1/750. If I subject the section to HIER, I can use the same primary ab at a dilution factor of at least 1/2000. Same applies to 90 C HIER vs M/W HIER: former requires 30 mins or more, latter requires ½ that time and, is more effective overall ( stronger signal/greater dilution factor).

Carl Hobbs FIBMS

Histology and Imaging Manager

Wolfson SPaRC, Guys Campus, London Bridge

Kings College London, London SE1 1UL

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