[Histonet] IF staining questions

[Eddie Martin]

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

NIH Bone Marrow Service 
RND clinical & anatomic pathology specialist

> On Oct 19, 2023, at 9:22 AM, Charles Riley <criley at udel.edu> wrote:
> 
> Why would one decide to use a primary antibody along with a secondary
> antibody rather than a primary antibody conjugated with the secondary?
> 
> Example.   I have a researcher who wants to do CD11C staining with
> Alexafluor488
> 
> Is it better to buy and use a primary antibody CD11C conjugated with
> Alexafluor488  or to do the CD11C primary and a Rabbit anti-rat (H_L) IgG
> antibody secondary?
>