Anti-SOX9 antibody (ab26414)

Inquiry: Antibody code: ab26414 Batch number: GR4041-1 General Information: Antibody storage conditions Aliquoted and stored at -20C Description of the problem No specific staining Sample Parrafin sections of both mouse and human testis Fixation of sample Paraformaldehyde 4% for 24 hours Antigen retrieval Heat induced, tried both citrate pH 6.0 and TE pH 9.0 Permeabilization step Blocking conditions Blocked in buffer containing 0.3% Triton, 0.05% Tween-20 and 4% Normal Donkey Serum. Blocker for 1 hour. Primary Antibody Diluted antibody in blocking buffer, incubated overnight at 4C. Washed with PBS. Secondary Antibody Biotin conjugated antibody from Jackson immunoresearch. Diluted 1:200 in blocking buffer. Detection method Cy2 or Cy3 conjugated avidin Positive and negative controls used Other antibodies stained the same sections with no problem. No negative control as the issue is lack of signal Optimization attempts (problem solving): How many times have you tried the IHC? 4 Have you run a "No Primary" control? No – again, not relevant. Do you obtain the same results every time? Yes What steps have you altered? I have altered the antigen retrieval buffers. Additional Notes Sox9 is a known marker for Sertoli cells in testis. Therefore, staining of paraffin sections of normal testis for Sox9 must show expressing cells. A different antibody that also stains these cells works fine, so the problem is not with the sample or slides. Thanks in advance.

ANSWER:

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab26414 Anti-SOX9 antibody. I would also appreciate if you can confirm some further details:

1. According to the questionnaire, the slides show a non-specific staining. I am wondering if the endogenous biotin has been blocked when you are working with a biotin-avidin amplification system as testis contains endogenous biotin?

2. Triton is quite a harsh detergent and usually only necessary when you want to permeabilize the tissue, but as a HIER was performed this should be obsolete. Triton can be quite harsh on some epitopes, therefore, you may increase the clarity of the staining by reducing the Triton concentration or skipping it altogether.


Should the suggestions not improve the results, please do let me know.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

Por favor, indiquenos unos anticuerpos validos para la siguiente situacion:
Applications: Immunochemestry
Reacts with: Chondrocytes or precursors of chondrocytes
Sample: canine (dog) trachea
Muchas gracias

ANSWER:

Gracias por contactarnos.

Varios marcadores pueden usarse para la detección de condrocitos mediante immunohistoquímica. Solo algunos de ellos se han testado en especie canina, el resto me temo que no se han testado, por lo que se desconoce su funcionamiento en ellas.

Le indico algunas posibilidades con los números de catálogo de Abcam:

Collagen II: podría usarse ab300 o ab34712. Ninguno de ellos se ha testado en perro. Si está interesado en usarlo en esta especie, quizás le interese participar en el sistema Abreviews de evaluación de anticuerpos, por el cual les ofrecemos un anticuerpo completamente gratuito al mandarnos los resultados de testar el anticuerpo en la nueva especie. Si le interesa, por favor, comuníquemelo antes de realizar el pedido.

Para saber más sobre el sistema Abreviews: http://www.abcam.com/abreviews

Aggrecan: ab3778. Tampoco testado en perro, pero susceptible de evaluar su aplicación.

CD29: ab30388. En este caso tampoco hay datos de su uso en especie canina, tendría que evaluarlo usted misma.

CD44: ab19622 y ab119863. Ninguno de ellos se ha testado en perro, sin embargo, debido al elevado porcentaje de homología con dicha especie, es probable que reaccione. Por ello, si le interesa comprobarlo, puede igualmente usarlo y enviarnos los resultados, beneficiándose de una futura compra totalmente gratuita.

SOX9: ab26414 y ab71762. En este caso, ambos anticuerpos se han usado y caracterizado en especie canina, por lo que están cubiertos por la garantía Abpromise.

Espero que esta información le sea de utilidad. Si necesita que le enviemos una proforma de alguno o varios de los anticuerpos, si necesita más información de alguno de ellos, o si quiere saber más acerca del sistema Abreview, por favor, no dude en volvernos a contactar.

Does this antibody cross react with SOX10?



Sincerely,

ANSWER:

Thank you for your enquiry.

I can confirm that the immunogen for ab26414 SOX9 antibody has 23% aligment to the human SOX 10 protein. This is very low, and so the antibody is not likely to crossreact wtih SOX10. However, I am sorry this has not specifically been tested in the laboratory.

I hope this information will be helpful to you. If you have any further quesitons, please do not hesitate to contact us.

This is giving a strong signal in cardiomysite, there should be weak or none. Do you know of any background issues with this product on cardiomyosytes or heart tissues in general?

ANSWER:

Thank you for contacting Abcam. I am sorry to hear of the difficulties that you have been experiencing with our product.

I have had an opportunity to discuss background issue with othe IHC experts here at Abcam. While each tissue type may present its own challenges we haveno knowledge ofa consensus regarding an issue in cardyomyosites.

I do hope that changing the fixation of the tissue will lead to better results. Please let me know if you have any questions.