My signals for immunofluorescence is very weak?

Question

is there any way to improve the positive signal? making it more intense on IHC slide staining

Answer 1

Have you tried titrating the amount of primary and secondary Abs you use, or are you just going with a recommended concentration?

For example, if the suppliers suggest using at 1:100 dilution, then you should try also at 1:50, or 1:25.

Do this for both the primary and secondary, and you'll hopefully settle on an optimum concentration for both.

Also remember to perform a positive control to make sure your antibody is in fact recognising what it should be (maybe there isn't very much target in your sample, for example)

Also - is it that your staining is weak, or do you have a high background? If the background is high, then try blocking with a higher concentration of serum before probing, or block for longer.

If your samples are formalin fixed, and you normally permeablise them with detergent, maybe you need to permeablise more thorougly (longer, or more concentrated detergent)?

Finally - how are your samples fixed? Some antibodies (usually monoclonals) won't work on formaldehyde-fixed samples, or won't work with methanol-fixed sections. Have you performed a positive control Western blot to determine if the antibody even works?