Scientific Forum

TM9 membrane protein

Hi BiCell

We’ve just received the result on our antibody, PO 361221.
The results in progress report (project 20210543 phase 3.pdf) seem to be very interesting, and promising I think.
Especially, the expression of TM9SF3 in the luminal membrane of the collecting duct seems very interesting.
We’d like to ask;
How do you know that it’s a collecting duct? , Is this because of the morphology?
Are all the positive cells collecting duct?
The lower cells show the staining on the plasma membrane and the right ones in the cytoplasm. Is that correct?

Then, we also would like to know the results on other tissues.
Could I ask you whether you have done IHC on all the tissues we’ve discussed?
If you have other results, we’d like to know.

best regards,
keisuke

Comments

  • June 16, 2022

    bicell-admin

    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

  • June 16, 2022

    bicell-admin

    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

  • June 16, 2022

    bicell-admin

    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

  • June 16, 2022

    bicell-admin

    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

  • June 16, 2022

    bicell-admin

    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

  • June 16, 2022

    bicell-admin

    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

  • June 16, 2022

    bicell-admin

    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

"I am really impressed with your approach. We tried multiple times previously to create monoclonal and polyclonal antibodies to claudin-2 and MLCK1. We have had limited success generating polyclonals and no success generating monoclonals. You have generated outstanding monoclonals to both. I look forward to continuing to work with you."

Jerrold R. Turner, M.D., Ph.D.

Brigham and Women’s Hospital | Harvard Medical School

"The polyclonal antibody you generated for KIAA0408 is stunning! KIAA0408 is a novel cilium molecule that has never been studied. So, clearly there will be a lot of demand for it as we have discovered a very interesting finding and the story will be published in a high impact journal. I am strongly inclined to generate monoclonal antibody for this protein too and we should think about patenting it."

Univ.-Prof. Jay Gopalakrishnan PhD

Heinrich-Heine-Universität | Universitätsklinikum Düsseldorf

"Your ARL13B antibody works beautifully!!! We’re so happy to have a cilia-specific antibody made in rat! I can send you high resolution images to be posted on your website."

Julie Craft Van De Weghe, PhD

School of Medicine | University of Washington

"The assay is a homophilic interaction mediated cell adhesion on purified protein (in this case, immobilized purified Pcdhga9 to Pcdhga9 expressed on cell surface). Compared to control, cell adhesion is reduced in the presence of Pcdhga9 monoclonal antibody supernatants!"

Divyesh Joshi, PhD

School of Medicine | Yale University

Contact us for questions or custom requests!