Hi Rania

I am using TMEM119 (1:1000) from abcam on free floating vibratome cut (60um) sections. I have to perform antigen retrieval by boiling the sections in citrate buffer (pH 6) at 80 C for 45 min (water bath). I have performed co staining with Iba1.

I think for frozen sections (if unfixed) you do not have to perform Ag retrieval. You can try mild fixation if desired before staining.

All the best!

Why not use an established IHC antibody for microglia (such as Iba1) from WAKO? It works great and has been used in countless publications.

Thanks for your input Tushar!

Eliot, I'm aware of the Wako Iba1, but Iba1 is not specific for microglia; it will stain infiltrating macrophages; my model is looking at inflammation in the brain and I would like to only look at brain-resident microglia and not infiltrating macrophages.

Hi Rania,

I used TMEM119 on mouse brain sections, 60 µm thick (nonfrozen though), free floating. I used 1:1000 dilution in 0.5% TritonX-100+5% goat serum, 1XPBS, 2 days incubation. It works great, no need to do antigen retrieval, at least for the tissue I used. I did, 2 days incubation because the first time I tried the antibody overnight, in 0.2 % TritonX-100, and saw no signal. So, I both increased the triton conc, I basically use the blocking buffer to dilute my primary antibody in, and extended the incubation time to 2 days overnight. It worked well, so I did not chnage anything again. You might try 1 day overnight and I am guessing you can even decease the dilution.

Good luck!

Thank you very much Gulcan!

Hi Gulcan and Tushar,

I tried staining with the Abcam TMEM119 (AB209064) antibody and unfortunately it did not go great. When I imaged my sections in confocal microscopy, I could only see green fluorescence everywhere (from the alexa 488 antibody I used) but no specific staining to any cell type anywhere. I'm wondering if you can let me know if there is something wrong with my protocol (which I adapted from the original Bennett et al. paper and Abcam's recommendations for their TMEM119 [28-3]antibody...

- Adult mice are PFA perfused and scarified, brains are post-fixed in 4% PFA at 4C overnight then moved to 30% sucrose-PBS solution to sink. Brains are cut on freezing microtome at 30 um thickness and tissue is collected in 6 well plate with PBS, stored in 4C fridge until staining. * I had waited about 3 weeks to stain my sections.

- Day 1 of staining:

1. Washed sections in PBS + 0.5% Triton X-100, 3 x 5 min washes.

2. Blocked sections with 5% blocking buffer (1X PBS, 0.5% Triton X-100, 5% Goat Normal Serum from Abcam) for 1 hour at RT on a shaker.

3. Added TMEM119 antibody (1 in 1000 dilution) and incubated in 4C fridge on shaker. TMEM119 antibody was diluted in 1% blocking buffer ( 1X PBS, 0.5% Triton X-100, 1% Goat Normal Serum from Abcam).

-Day 2 of staining:

4. Washed sections in PBS + 0.5% Triton X-100, 3x 5 min washes.

Performed steps 5- 8 in Dark (covered 6 well plate with foil):

5. Added secondary Alexa Fluor 488 antibody ( 1 in 500 dilution)for 2 hr at RT on shaker. Secondary antibody was diluted in PBS only.

6. Washed sections in PBS only, 3 x 5 min washes.

7. Mounted sections and coversliped with Vectashiled Mounting Media with DAPI.

I would greatly appreciate any advice you can share.

Thanks,

Rania

Hi Rania,

A probable solution could be: Antigen retrieval. Abcam does recommend it, please check. I keep the sections in pH6 Citrate buffer in an eppendorf tube and incubate at 80 C in a water bath for 45 min, before blocking.

You can try other antigen retrieval methods as well.

I dont see problem with any other of your steps.

A longer incubation as suggested by Gulcan might work but I have no experience with it.

Best wishes,

Tushar

Rania,

I did not freeze the brain after PFA fix. I also incubated the sections with primary antibody for two days in cold room.

I hope it helps..

gulcan

We are having similar issues with background in human PEG and human/mouse paraffin using citrate or EDTA retrieval at various concentrations of TMEM119, may try a longer incubation. Open to any other suggestions.

J

This is how my staining with TMEM119 ( AB209064 ) turned out following the protocol I wrote up above...

Gulcan, thanks for your clarifications. I am thinking of going for a longer incubation period (2 days instead of 1 day overnight) with the primary.

Hi

We recently used this antibody. Although we are still optimizing the concentration, staining is pretty good. This antibody majorly stains the microglial processes (See attached image).

Staining details: Mice brains (8 weeks), PFA perfused, TMEM119 (1:300)