I'm working with 40um fresh frozen (non-perfused) sections of rat brain tissue and I'm staining for wisteria lection (immunofluorescence). After staining, my tissue has high background and a weak signal for lectin. I'm wondering if this happened because the blocking solution ran off the tissue during incubation. Before blocking, I fix my sections in 4% PFA for 10 mins and then do 3x 5 minute PBS washes. Because the slides are still a little wet, I think that's causing the blocking solution to run off. If this is what's happening, could that account for high background and a weak antibody signal?
Forget the problem of blocking, that will not matter. What is the source of your second antibody? If it is from rat, you could change it to rabbit or goat. Then the high background problem will be solved. You can also try to change the dilution of of your primary antibody to 1:200 or even 1:50 to get a stronger signal.
Yes, improper blocking can result in high background and weak target signal. The section needs to be level and in good contact with the blocking buffer throughout incubation. You can increase washing till you think the fixative has been completely washed off before blocking.
Hi Marcia,
is there any reason you need fresh frozen tissue rather than transcardially pfa fixed one? I did a lot of stainings with both WFA and VVA lectins on fixed tissue and floating sections. Normally I stain 3 (mouse brain) sections in a well of a 24-well plate in at least 500 ul volume.. Never let the sections drying out...
Federica Filice , I'm glad you asked. If given the choice, I would almost always choose perfused tissue. The brains I'm working with were collected at my PI's previous institution where she planned to do an autoradiography project with them. I guess they need to be fresh for that type of processing. Due to budget constraints and COVID, these brains are what I have to work with now for an IHC project now. I piloted this stain on these tissues beforehand and the stain looked great. My guess is that during big IHC runs, there's less time to tap off excess PBS befor blocking. I'm going to try submerging the slides in blocking solution and see how the stain comes out.
Praveesh Valissery , I think you're right. I bought a leveler a while back and placed it on top of the staining trays to make sure they were level and not an angle. I think what I need to do is submerge my slides in blocking solution so I don't have the run off problem. If I try to tap off all the excess PBS, I'll risk the slides drying out. I'm processing around 200 slides in each IHC run.
Hi, Xin Ren. I'm using goat serum, but both my primary (Wisteria Lectin) and secondary (Strep488) antibodies are not raised in an animal, not that I'm aware of. During piloting, I got minimal background and great signal on fresh rat tissue after it was post fixed in PFA. There's something going on between piloting and running my cohort of slides that's causing a problem.
Slides drying out in between might cause high non-specific background. Because you did not mention is: Do you use a hydrophobic pen (e.g. "DAKO pen" or "PAP pen")?
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