Anti-GATA3 antibody (ab32858)

Inquiry: No staining was seen. Detailed IHC protocol, photos of stainings and user information are on the attached file.

Antibody information:

Antibody Name: GATA3
Antibody code number: ab32858
Antibody lot number: GR 58081-1

How is the product stored ? -20 °C (deep freeze)
Complaint: No staining was seen (You can find the photos of our stainings at the and of the form)
Positive control tissue: Tonsil and spleen
Which tissue and from which organism was used: Paraffin embedded human tissue
Fixative and Fixation time: Formaldehyde 12-24 hours
IHC Detection Kit brand, name, code number:
On Ventana Benchmark XT otomatic IHC staining instrument,
“ultraView Dab Detection kit” ( Roche code: 05269806001)

DETAILED IHC PROTOCOL:
Pretreatment (citrate? EDTA? Or another?): EDTA, 60 minutes on the instrument
Protein blocking incubation time: No protein block
Primary Antibody dilution and incubation time and temparature: 1:100 dilution. 32 minutes incubation at 37 °C

Secondary antibody dilution and incubation time:

RTU Secondary antibody from “ultraView Dab Detection kit” ( Roche code: 05269806001)- HRP conjugated

8 minutes incubation on the instrument


Sterptavidin HRP incubation time:
No Sterptavidin HRP; because the kit is multimer technology based (secondary HRP conjugated)

Chromogen (DAB? AEC? Or another?) incubation time:

DAB from ultraView Dab Detection kit;

8 minutes incubation on the instrument


Additional application:
It is used a inhibitor from the kit before primary antibody. Inhibitor incubation time is 4 minutes.

ANSWER:

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

I would like to reassure you that ab32858 is tested and covered by our 6 month guarantee for use in IHC-P and in human and mouse samples. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.

Reviewing this case, I would like to offer some suggestions to consider which I hope wouldhelp optimize the results. I would also appreciate if you can confirm some further details:

1. Please confirm which species has been tested.

2. Antigen retrieval can sometimes require individual optimization. I can recommend to Try citrate buffer and different time points (2, 5, 10, 15 and 20 minutes).The Abreview published on the datasheet submitted by one of our collaborators has used citrate buffer retrieval successfully, so I can suggest to try this.

3. I can recommend to try overnight incubation 4oC. This can often provide more specific and efficient staining.

4. As GATA3 is a nuclear protein,I can recommend it would be important to include a permeabilization step. Try incubating in 0.2% Triton in PBS for 10 minutes. I can also suggest to include 0.2% Tween, a more gentle detergent,in the wash buffer and antibody dilution buffer afterwards. This will help to keep the antibody solubilised and the cells permeabilized.

5. I am sorry I am not sure what is meant by 'It is used a inhibitor from the kit before primary antibody. Inhibitor incubation time is 4 minutes'.Could you explain what inhibitor has been used, and why?

6. I can suggest to increase incubation time for secondary antibody to help increase the staining. Has this worked well with other primary antibodies?

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

I would like to use anti-GATA3 antibody for double immunoflouresecnce staining of mouse frozen sections.  Checking the web, I noticed that abcam has several antibodies for GATA3.  Which one would you recommend?   Thank you.

ANSWER:

Thank you for contacting us.

I have checked our catalog and found 4 anti-GATA3 antibodies that have been tested in mouse (ab32858, ab106625, ab82828 and ab110093), however none of these antibodies has been tested so far in frozen tissue sections (IHC-Fr).

ab32858 and ab106625 were tested so far in IHC-P (paraffin sections) and WB.

ab82828 has been tested in ELISA and WB

ab110093 has been tested in ICC/IF and WB

Any of the IHC-P or ICC/IF tested antibodies are worthwile to try with frozen sections.

For UNTESTED species and/or applications, we have established a testing discount program. Here is a brief description of how it works:

The testing discount program is for customers who like to use an antibody/protein on an untested species/application. You would purchase the antibody at full price, test it and submit an Abreview with your data (positive or negative). On your next order you will receive a discount for ONE antibody at the full price (100%) of the antibody you have tested. The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

Please let me know if I shall send you a discount code for trying out one of these antibodies.

I look forward to hear back from you and assist you further.