TM9 membrane protein

Hi Dr Sako,

Tm9sf3 is very bright in the kidney. Its expression in smooth muscle is relatively dimmer. I am sure you will have fantastic results from kidney of WT and KO mice. In the kidney, we can see a set of tubules extending from medulla to cortex. In cortex of sagittal tubules, we can see tubules with positive cells and negative cells intercalated with each other. Transverse section is not very indictive of this feature. There is almost no doubt it is collecting duct. From the pattern, it is also principal cells. The two cells showing cytoplasm is most likely related to how cells are orientated. The luminal localization also extends to sub-apical regions of cytoplasm.

For co-staining, please order this two products:
https://bicellscientific.com/product/aquaporin-2-antibody/
https://bicellscientific.com/product/pendrin-antibody/

For ion channels, we generally recommend 2% PFA fixation. Our protocol is here:
https://bicellscientific.com/education/article/protocol-for-staining-pendrin/

Since Tm9sf3 results are very good, we didn’t look through many other tissues. Mainly epithelial tissues, such as colon and liver. There is no expression of Tm9sf3 in mucosal epithelium or hepatocytes.

BiCell team

Hi BiCell,

I have tested the kidney samples from WT and KO.
The age is E18.5 because KO dies after the delivery.
I sacrificed the mother, then removed the kidney from an embryo.
The samples were fixed in 4% PFA/PBS for O/N.
After fixation, samples were rinsed with PBS, then 30% sucrose.
Samples were sectioned into 10um slices by cryostat, then stained.
I used 1/100 dilution of primary Ab.
The samples showed a pretty similar staining pattern, some cells/tissue showed a bit strong signal.
I haven’t seen any difference between WT and KO.
Could I ask for some advice?

best regards,
Keisuke

Hi, Dr. Sako,

Tm9sf3 was stained in P1 pup (tm9sf3-embryo-image-bicell.pdf). We can see a similar pattern to adult mouse kidney but at a reduced expression level. The reason could be either low expression before kidney maturation or poor fixation, since we cannot perfuse the embryo/pup.

BiCell team

Hi BiCell

Thank you for your images.
I have a question about the images you sent to me.
When I did staining on cryosectioned E18.5 kidney using the Ab from Bicell and Atlas antibodies,
I found the staining of TM9SF3 in the intracellular organelle, perhaps the Golgi, but not on the plasma membrane (tm9sf3-embryonic-kidney-211109.pdf).
Still, I’m doubting the results, but the difference between WT and KO are obvious.
In your images, only a couple of cells are positive for TM9SF3 staining.
I did antigen retrieval with boiling in citrate buffer or 1% SDS.
Of course, this could be a cause, but it can not explain the difference.
How do you think about it?

best regards,
Keisuke

Hi, Dr. Sako,

I see what you mean. It appears that neither antibody is working, because neither is giving a pattern. The cytoplasm reinforcement (slide 1 and 2) may well because those tubules are thicker. Microtome or cryostat sections are not always smooth. Slide 3, KO comparison (Atlas) isn’t because the specific binding is lost, but sections are from two different animals, and drop fix can create a lot of variation from sample to sample.

I am suspecting that fixation is a major problem. Drop fix is alway poor compared to perfusion. I would recommend to perfuse an adult animal and stain the kidney with both our antibody and Atlas. I am sure you will start to see pattern. Ion channels on kidney tubules always show pattern, which indicates a cell biology of the protein, e.g. plasma membrane, brush border, et al.

Meanwhile, I would also recommend to stain with these two antibodies on both adult kidney and E18 kidney. I am sure you will see how fixation will impact signals. These two antibodies are well established and tested by many labs:
https://bicellscientific.com/product/lrp2-antibody/
https://bicellscientific.com/product/aquaporin-2-antibody/

BiCell team

Hi BiCell,

We’ve almost completed the validation of our TM9sf3 antibodies.
When I tested 2% PFA fixation on zebrafish embryos, I managed to detect the TM9SF3 signal on the plasma membrane (anti-tm9sf3-staining-on-zebrafish-embryo.pdf).
I attached the file, so please have a look.
Still, the signal is not perfect, so if you have any comments, that could be helpful for me.
I think the incubation time of 2nd antibody would be too short.

Anyway, I think now it’s time to proceed with our process.
Is antibody purification the next?
If so, could you tell me the timeline of the process?
Also still I’d like to test your electron microscopy grade PFA.
Is it possible to include it when you ship antibodies to us?

Best regards,
Keisuke

Dear Keisuke,

Very nice work! I agree, if for whole mount staining, then please do incubation of primary and 2nd antibodies sequentially at 4oC for overnight.

If embryo is not too fragile, then you could try to increase PFA to 3.7% and slowly rotate the sample during overnight fixation on a belly dancer.

Since whole mount works, cryostat sections will work for sure. For cryosection, after fixation, please add a 30% sucrose protection step to improve the tissue morphology on cryostat. Please prepare 30% sucrose in PBS, not water.

Please ask Nacalai to arrange for shipment. It will take us a week to purify all 4 antibodies and we can ship them to Nacalai next week.

Yes, absolutely, we will send you EM grade PFA.

BiCell team

You must be logged in to add new comments. Please log in by clicking the link below:
Register or Login