Pax7 Immunofluorescence

The paired homeobox protein 7 (Pax7) is a widely used marker of satellite cells, with the most commonly used antibody for this stain the goat anti-mouse ab produced at the Developmental Studies Hybridoma Bank (DSHB). To get a good stain, while limiting non-specific staining, requires the use of either an antigen-retrieval step, or kit designed for mouse antibody staining of mouse tissue (or a combination). I have found that both the protocol described here, and the M.O.M. kit available from VectorLabs give good results. The protocol listed below is modified from a protocol by Dr. Xue-song Feng.

Protocol

  • Remove slides from -80C freezer and allow to equilibrate to room temperature
  • Briefly fix the sections in 4% PFA for 10mins
  • Rehydrate the sections in PBS+0.1% Triton-X 100 (PBST), for 2x5mins
  • Perform heat activated antigen retrieval by placing the slides in pH 6.0 citrate buffer (from Invitrogen) and heating in a high pressure cooker for 10mins (we use this model cooker)
  • Allow the slides to return to RT
  • Wash in PBST for 2x5mins
  • Block sections in PBST+3% BSA + 10% Affinipure FAB goat anti-mouse IgG for 45mins
  • Wash in PBST 3x5mins
  • Apply primary antibody/s:- Pax7, diluted 1:10 in PBST+3% BSA + 5% normal goat serum (other antibodies at appropriate dilution)
  • Incubate overnight at 4°C
  • Wash in PBST for 3x5mins
  • Apply secondary antibody/s diluted in PBST + 3% BSA and incubate for 60mins (Goat anti-mouse IgG1 for Pax7)
  • Wash in PBST with Dapi for 5mins
  • Wash in PBST for 2x5mins
  • Mount slides with VectaShield mounting medium and coverslip

Figure. Cross-section taken through the gastrocnemius muscle of a 7 day old mouse and stained for myosin (bottom left, MF20-green), Pax7 (top right, Red), and nuclei (top left, dapi-blue). A merged image is provided in the bottom right, where it is possible to see the ‘pink’ satellite cells, located at the edges of the muscle fibers.

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9 thoughts on “Pax7 Immunofluorescence

  1. Yi Fang

    Hi James,
    i have a question about Pax7 western blot. i have done Pax7 WB by DSHB antibody in injured and uninjured TA muscle ,but there were many bands around 55kDa, i couldn’t distinguish which band is PAX7.. In addition, Pax7 or MyoD is partly merge with DAPI in tissue Immunofluorescence, i dont know it is a false positive .So, could you give me some advice about it
    best regards,
    Yi Fang

    1. Alicia

      The problem you are having with multiple bands on a western is the result of denatured immunoglobulin present in your muscle tissue. This will always show up at the heavy chain and light chain sizes of 55 and ~30kD when using any anti- mouse secondary antibody on mouse tissue- derived lysate. People doing immunoprecipitation and westerns have the same issue. Since pax7 is around 55kD, the heavy chain will mask it. You can get around this problem using a light chain specific anti mouse secondary antibody. Then only the light chain nonspecific band around 30kD would show up, in addition to the pax7 band. Think of the mouse on mouse problem with sections. This is the same problem for westerns.

  2. Yi Fang

    Hi James,
    i have a question about Pax7 western blot. i have done Pax7 WB by DSHB antibody in injured and uninjured TA muscle ,but there were many bands around 55kDa, i couldn’t distinguish which band is PAX7.. In addition, Pax7 or MyoD is partly merge with DAPI in tissue Immunofluorescence, i dont know it is a false positive .So, could you give me some advice about it
    best regards,

    1. Dear Yi,
      You should be able to get a single band for Pax7 in an injured TA (use day 3-5 as a positive control) via WB. Use the DSHB Pax7 (supernatent) antibody at a dilution of 1:100.

      For the Immunofluorescence, you should see clear overlap between Pax7 and Dapi (although, it is excluded from heterochromatin).

      James

  3. Bo

    Hi James,

    I just want to confirm that I am looking at the right antibody from DSHB, a link that you listed above. It seems like the antibody on that link is mouse anti-chicken Pax7 instead of goat anti-mouse. Is this right one? Also, you recommend Pax7 (supernatent) antibody, correct?
    Also, with the protocol you recommended, will MF20 work as well?

    Thank you so much.

    Best,

    Bo

    1. Yes, the link is the correct one (my apologies). I use the supernatent version, but you can use the concentrate (just dilute accordingly). Also, the MF20 antibody works perfectly with this technique.

  4. Bo

    Thank you so much for your quick response. I just wanted to check with you.
    One more questions though, are there any other markers or staining that we could do on the frozen but fixed tissue section to detect or differentiate different muscle fiber types?
    Please understand that I have so many questions as I am a pretty new in this field.

    Thank you.

    Bo

  5. Yi

    Dear Dr.James,

    I have a few questions about your Pax7 IHC protocol.
    1.what is the temperature of antigen retrieval in a high pressure cooker?
    2.you showed the tissue picture of a 7 day old mouse.whether this protocol is also available to older mice(P60 or elder)? Is there any condition to chang or replace?
    3.i have tried muscle embedding,but there are still many holes in my muscle cross-section.whether i should take it to PFA fixtion and/or sucrose dehydration before?if so,whether it would influence IF in mouse tissue.Could you give me some suggestions?

    In addiction, as a freshman in muscle biology,i often meet many technical problems about muscle methods.Could you provide me your email address,and i could ask you questions more conveniently.

    i am looking forward to hearing from you.
    best regards,
    Yi

    1. Hi Yi,

      To answer your questions:
      1. I’m not sure what the temperature is inside the pressure cooker. It will be above boiling to generate the steam needed, but beyond that I’m not sure.
      2. The technique will work for tissue isolated from mice of all ages. Just be aware that the number of satellite cells will be reduced in adult mice.
      3. I would suggest you follow the technique I have described for tissue fixation (fresh tissue into low concentration PFA).

      I’m happy to give comments and help out on this website when I can, but your first port-of-call should be your lab head. As I run my own research program (with my own students 😀) I can’t always make time to answer technical questions.

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