Anti-FHIT antibody (ab53074)

Customer has asked

how to do the antigen retrival by which method are u advising microwave or pressure cooker??????????????and how many cycles we need to repeat by microwave

Regards

ANSWER:

Thank you for your reply.

I am pleased to provide the testing protocol below, including the antigen retreival. I hope this will be helpful. Please note this is a guideline only, and may require some further individual optimization by the end user.

I look forward to hearing from you with the results of the next experiment and hope we can resolve this case as soon as possible for your customer.

PROTOCOL:

Deparaffinization

1. Incubate slide at 60℃ for 60 minutes.
2. Deparaffinize in Xylene for 10 minutes and repeat one more times.
3. Hydrate in 100% alcohol for 5 minutes, in 95% alcohol for 5 minutes, in 85% alcohol for 5 minutes, in 75% alcohol for 5 minutes.
4. Dip into Distill Water for 5 minutes.
5. Dip into TBS (50 mM Tris, 100 mM NaCl, pH 7.6), leave for 5 minutes, and repeat two times.

Antigen Retrieval

6. Bring 500 - 2000 ml 10 mM citrate buffer (pH6.0) to the boil in a stainless steel pressure cooker.
7. Put the slide into staining rack and lower into pressure cooker ensuring the slide is well immersed in citrate buffer.
8. When the pressure indicator valve has risen after 3-4 minutes, incubate for 1 minute.
9. Cool the slide naturally to room temperature.
10. Dip into distilled water, leave for 5 minutes, and repeat two times.
11. Dip the slide in TBS for 5 minutes and repeat two times.
12. Immerse slides in 3% H2O2 (in fresh methanol) for 15 minutes at room temperature.
13. Wash with distilled water two times, 5 minutes each time.
14. Wash with TBS (pH 7.6) two times, 5 minutes each time.

Staining with Primary Antibody

15. Dilute primary antibody with 3% BSA in TBS. Cover the tissue section on the slide with diluted primary antibody (use 50 – 150μl for each slide).
16. Incubate at 37℃ for 30 minutes or at room temperature for 60 minutes (The optimal incubation time, incubation temperature, and antibody dilution should be determined by the individual laboratory).
17. Wash with TBS two times, 5 minutes each time.

Staining with Secondary Antibody

18. Incubate with 100-200μl Polymer Enhancer. Incubate 30 minutes at 37℃.
19. Wash with TBS for 3 times, 5 minutes each time.
20. Incubate with 100-200μl Polymerized HRP and incubate 30 minutes at 37℃
21. Wash with TBS for 3 times, 5 minutes each time.
22. Add DAB solution and incubate 3-10 minutes(The reaction progress and the optimal time should be determine according to microscope).
23. Wash with distilled water for 2 times, 5 minutes each time.
24. Counterstain sections in hematoxylin if required,wash with distilled water.Immerse slides in 0.1% HCl- ethanol for 1-10 seconds, wash with distilled water.
25. Dehydrate through 95% ethanol for 1 minute, 100% ethanol for 2×3min, Xylene for 2×3min, and coverslip with mounting medium.

DESCRIPTION OF THE PROBLEM No staining
SAMPLE freshly formalin fixed paraffin embedded tissue sections

PRIMARY ANTIBODY Concentration or dilution 1100.150 and 125 Diluent buffer PBS and diluent Incubation time 1hr,1 and half ,2hr,and 2 and half hour is tried Incubation temperature room temprature
POSITIVE AND NEGATIVE CONTROLS USED as prescribed by the company that is breast carcinoma and squamous cell carcinoma
ANTIBODY STORAGE CONDITIONS as mention in the form
FIXATION OF SAMPLE Fixation: Yes/No Fixative agent and concentration Fixation time Fixation temperature
ANTIGEN RETRIEVAL both microwave and pressure cooker has been tried
PERMEABILIZATION STEP Yes/No Permeabilization agent and concentration Permeabilization time Permeabilization temperature
BLOCKING CONDITIONS Yes/No Blocking agent and concentration Blocking time Blocking temperature rom temp Endogenous peroxidases blocked? Yes/No Endogenous biotins blocked? Yes/No
SECONDARY ANTIBODY let me collect from customer
HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 5
DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes
WHAT STEPS HAVE YOU ALTERED? all most all the possible alteration we have tried

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 ab### is tested and covered by our 6 month guarantee for use in IHC-P and human 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 help optimise the results. I would also appreciate if you can confirm some further details:

1. I would appreciate if you are able to provide an image which would help me to assess the results.

2. Please confirm which buffer was used for antigen retrieval? I can recommend to try citrate buffer if not already done. Trying different time points can also help optimize the results, for example 2, 5, 10 and 20 minutes.

3. As this is a cytoplasmic protein, I can recommend to include a permeabilization step. For example, try 0.2% Triton for 10 minutes.

4. I am sorry, the dilution information is not clear for me. For IHC-P with this antibody, we recommend a dilution of 1/50 - 1/100. I can also suggest to try incubating overnight 4oC which can often provide more efficient and specific staining.

5. Please provide information on the fixation as requested in the form.

6. Please provide information on the blocking step as requested in the form.

7. Please provide information on secondary antibody. Is the current vial of secondary antibody working well with other primary antibodies?

8. Please provide further information on the alterations and optimization that have already been tried.

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.

LOT NUMBER GR35255-1 ORDER NUMBER 1023530 DESCRIPTION OF THE PROBLEM No staining SAMPLE Species Human Type of sample: PFA/formalin fixed paraffin embedded sections PRIMARY ANTIBODY Concentration or dilution : 1:50, 1:100, 1:200 Diluent buffer : Leica dilution buffer Incubation time : 15min, 1hr, 2hr, overnight Wash Buffer : Bond Wash Solution Number of washes: AR9590 DETECTION METHOD Compact Polymer method POSITIVE AND NEGATIVE CONTROLS USED Positive control : Breast Carcinoma ANTIBODY STORAGE CONDITIONS -20℃ FIXATION OF SAMPLE Fixative agent and concentration : 10% Formalin Fixation time : 8Hr Fixation temperature : RT ANTIGEN RETRIEVAL pH 9.0 Tris-EDTA buffer & pH 6.0 citrate buffer PERMEABILIZATION STEP - BLOCKING CONDITIONS Endogenous peroxidases blocked? Yes SECONDARY ANTIBODY Universal (DS9800, DS9958)Mouse & Rabbit IgG Concentration or dilution: Ready to Use Incubation time : 10min, 15min, 20min Wash Buffer : Bond Wash Solution Number of washes: AR9590 HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 10 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? - ADDITIONAL NOTES -

ANSWER:

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

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

Reviewing this case, I would like to offer some suggestions to help optimise the results from ab53074.

1. ) I suggest to use a mild permeabilisation agent in all puffers. FHIT is cytoplasmic and therefore 0.1% tween might be the right choice.

2.) I suggest to incubate the primary antibody over night at 4C without any blocking agent in PBS only.

3. ) I also recommend to run a timecourse for the antigen retrieval step. This step always has to be optimised.

I would also appreciate if your customer can confirm some further details:

1.) For how long was the antigen retrieval performed?

We are happy to offer this technical support. In the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

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

Dear technical support team: This customer raised an enquire about ab53074 (Anti-FHIT antibody), this customer wants to know the immunogen sequence of this antibody or the range of this synthetic peptide (e.g. 20~50 amino acid), could you please help this customer to solve the problem? Thanks for your kindly assistance.

ANSWER:

Thank you for your patient.

The immunogen peptide sequence range is amino acids 110-130.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.