I have tried 4 IBA1 antibodies and cannot seem to get good staining. Mice were perfused/fixed with PBS/4%PFA. Brains were placed in PFA overnight, and then moved to 30% sucrose and frozen in OCT after sinking. My sections typically tend to be thicker (30um or 35um), but we do sections on slides instead of free-floating due to less handling and integrity of the structures. All of my antibodies work except for these IBA1 antibodies. I have tried permeabilizing with triton and saponin and got similar results. (Fix for 5 minutes on slide with 2% PFA, perm with 0.3% triton 15 min, block with 10% goat serum 30 minutes, then primary and secondary incubations with blocking serum). Antibodies are spun before addition.
Can anyone advise me as to why these IBA1 antibodies are creating so much background at both 1:100 and 1:1000, and why it is not staining the filaments of the microglia? Any advice is greatly appreciated.
Have you tried the Wako Iba1 antibody (rabbit)? Most people I know use this antibody with no problems. Your original protocol looks right to me, although with 30 um sections, I think free floating would give you better results (more penetration from all sides). I can't open the image, but based on your tags I assume its for immunofluorescence? Reducing the concentration of the secondary (or even the primary) antibody may reduce background. Also, cutting thinner sections might be better for IF, but if you want to see all of the branching of a single microglia, 30 um is a good thickness.
Hi Lauryn Evans
I would recommend you to use Anti Iba1 Antibody from FujiFilm Wako.
Please find the link attached.
https://labchem-wako.fujifilm.com/europe/category/01213.html
Thanks,
Nicole Schartz Nicole, thank you so much for your reply. That Wako IBA1 antibody you mentioned, do you know the catalog number? I am going to ask my PI to try it out.
Hi Lauryn,
I agree with Nicole Schartz, your protocol seems good and you should have a good staining with it.
I don't know which references you used, but I used Abcam and Wako antibodies in IHF on paraffin and frozen sections, and it works very well.
Here is the reference of the Wako antibody: Fujifilm (Cellular dynamics), #019-19741.
I'm sorry I can't help you more. I hope it will work with this one.
Best,
Anne-Sophie
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