We are attempting to label microglial cells in rat brain slices, however, facing some troubles with Iba-1. Does anyone suggest another specific marker for rat microglial cells? Is there a marker like TMEM119 specific for rats?
Hi Dirson,
I am assuming that you don't really mean "specific for rats" (as in selects rat microglia over other microglia) but rather a microglial-specific marker that works in rats.
As alternatives to visualize microglia in slices, you could try IHC/IF against Cd11b, CX3CR1 or P2RY12, or use one of the older/cheaper methods that exploit glycoprotein/carbohydrate binding (think tomato lectins among others) or NDPase histochemistry.
And lastly, you could also try to optimize your Iba1 staining. I personally do not care much for it as a marker of morphology or "activation", but it is definitely one of the most widely used stains in the field. This means you can definitely find reagents and protocols that will work for you, you just have to look around and try a lot!
Good luck!
Best,
Alberto
Hi Alberto,
Yes, I was meaning "microglial-specific marker that works in rats".
Thanks for your helpful answer.
Best,
Dirson
Very unusual not to get good labelling in the rat using Iba-1. Would be inclined to look very closely at your fixation conditions. How long do you post-fix the tissue for?
Could you provide more info on what antibody that you have used and the tissue conditions; we might be able to help more with getting the IBA1 to work.
:-)
I have gotten good staining using Wako Cat# 019-10741 Anti-IBA1, Rabbit in rat tissue.
Indeed, the WAKO ab is lovely. You may wish to perform a brief antigen-retrieval step prior (my go-to is a Tris/tween at a pH of 8.5 for 20 m @ 85 degrees; this allows one to use the ab above at very reasonable dilutions from 1:1000 to 1:5000 depending upon tissue thickness, etc. Some recommend Triton as well, but the AR is superior in my hands.
One note: I've noticed I have much better luck when storing the aliquots of WAKOs IBA-1 if the freezer is kept at -20. I kept it in a -40 and lost almost all staining--likely due to structural damage of the reagent.
It is the critical issues that there is no specific marker for the microglia (and for some other immune cells too), but Iba-1 works nicely to identify microglia (also the markers for peripheral macrophages). It is unusual that you faced some problem with it. Importantly, there is no clear description of the problems in your question too. Wako Iba1 should work nicely. There might be some issues in the protocols you are using. You can get it (protocol for Iba1 IHC) elsewhere though. For example, you can go through PMIDs: 25789481; 28178289; 28343295; 30305119 etc. Good luck.
Iba-1 antibody from Wako seems to work well to identify microglia in rat brain, at least in my case （with a dilution of 1:100）. You may also try OX-42.
I noticed that Iba-1 staining are slightly more sensitive to perfusion/fixation issues respect to anti p2y12R stainings in mice (it worked in the worst tissue). Both are basically microglia-specific, the labeling is different though, while Iba1 is strong in the nucleus, p2y12 membrane receptor is labeling especially the distal processes. Is way more challenging for automated counting of cells, but is pefect for morphological studies. If u have pro-inflammatory conditions P2Y12 gets often downregulated, so depending on ur conditions u can have a more or less clear staining. Good luck
No idea if you are still looking for this, but since it's on ResearchGate, eventually someone will be looking. I found this option: https://www.sysy.com/products/tmem119/facts-400211.php
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