Hi Sayaka, I have used ImmPact Red AP substrate kit for immunohistochemistry on several occasions with no particular problems, apart from one occasion with a very special material when the antigen was very damaged. ImmPact Red can be observed with bright-field microscopy or fluorescent - I have switched to fluorescent and had much better microphotographs - sorry, not very helpful in your situation. Years ago I have used New Fuchsin substrate for AP and it was exceptionally good, but the newer versions of NF were easily affected by organic solvents. OriGene had an "AP-Red+" kit, maybe worth checking if they, or another reseller have it.

Good luck with your project.

Hi Sayaka - I have recently been using ImmPACT Vector red for immunohistochemistry in human brain sections and found it to be very region specific in its sensitivity. Superficial cortical regions are coming out quite lovely but more interior structures are devoid of quantifiable cells. This does not appear to correlate with the primary antibody's affinity as swapping detection substrates gives us the expected number of cells across regions. The tissue I am working with is very heavily fixed and that might account for some of the gradient and may not apply in your case.

Are you adding an avidin biotin complex (ABC) prior to developing? If the answer is yes, are you sure it isn't expired or mixed incorrectly? No background to me means that the complex was either skipped or is not working right.