We are looking for a good alternative to the Rabbit-Anti-Iba1 from Wako as although it it great it limits the use of other rabbit antibodies for double labelling. Does anyone have any experience with this Goat-Anti-Ibal antibody from Abcam?
We have not tried this antibody from Abcam but the monoclonal Anti-iba1from Millipore (MABN92) and single staining works OK but the signal in mouse brain is weaker that those with the Rabbit Anti-iba1 from Wako, even incubating for 2 days. In addition we need to use the pre-incubation with Fab Fragment in order to avoid background staining
I have experience with it in the mouse. We tried to replicate our Wako protocol and it did not work. We did some troubleshooting and through in the towel and went back to Wako's anti-IBA-1. We got some very light label, even with Immpact DAB. Wako's just seemed to work for us better and was worth the extra money.
We have used the Wako rabbit too, but preconjugated it with a Zenon kit (molecular probes). It seems to work ok. We have also tried other prelabelling kits with similar results.
We have tried this goat-anti iba1 (Abcam) antibody on mouse brain tissue. Although definitely not as good (weaker) as the wako antibody, it works on free-floating 30 µm thick sections without any pre-incubation.
We have both and have found the Abcam goat antibody to be inferior to the Wako rabbit antibody.
Yes, this is a beautiful antibody, the dilution is 1;200 and work better if antigen retrieval is perform. The secondary, we use sigma Cy3 conjugated antibodies.
Thanks everyone for your responses ! @Eliseo, could you please tell me the tissue type, species, and method of fixation you have used with the abcam antibody? We have used the wako in brain tissue from pig/mouse/sheep/human/monkey as PFA fixed and paraffin or frozen but I wish to use the Abcam in brain from monkey/mouse paraffin embedded for double labelling. Thanks again !!
Bobbi- I use 4% PFA fixed PEG-embedded mouse brain tissue and I get good labeling with the Wako. I do not get good labeling with the Abcam, but I can share my protocol with you. I ended up having to use different antibody titrations and retrieval steps when I tried to switch from Wako to Abcam.
Kimberly, that would be good - Thanks. Wako is so easy and clean, but the double labeling is an issue that I need to get around and so I will give it a go and let everyone know how it goes.
Hi Bobbi,
Probably a bit late now... One of the most robust antibodies I have ever used.
Deeply anaesthetize and transcardially perfuse mice with ice-cold 0.9% saline followed by 4% paraformaldehyde. Remove brains, postfix in 4% PFA for 24 h at 4 °C, then cryoprotect in 30% sucrose for a minimum of 3 days. Snap freeze brains on liquid nitrogen and section on a cryostat. Store sections in cryoprotectant solution (25% ethylene glycol, 25% glycerine, in 0.1 M phosphate buffer) at –20°C.
Staining protocol is pretty standard but I find antigen retrieval (10mM Citrate buffer (pH9) pretreatment, 80 oC 30 mins) necessary for best results. Primary antibody concentration of 1:500 ( 4 °C, O/N), followed by secondaries at 1:1000 for 2 hours at room temp. Results possibly enhanced when using CF-dye conjugated secondaries.
Over and out.
Pete
Hi!
I 'm looking for a good mouse-Anti-Iba1 Ab for double labelling (HIC on fixed tissue of mouse) . Could someone suggest me which one to choose to get the best result? I used rb-anti-IBA-1 from Wako with antigen retrieval ant the staining is ok.
Thank
Michela
Hi Bobbi!
I have the same problems as you, but I can´t used Wako, so I have to figure it out somehow..
My biggest problem is the background. I tried different blocking solutions and it isn´t working out.
Any help will be welcome :) Thank you in advance!!
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