Anti-TRAP/CD40L 抗体 (ab65854)

Thank you once again for your follow up email.

I am pleased to let you know I have now heard back from the originator with a copy of the general IHC-P protocol that they use for testing. Please note this will be a guideline only and may require some further optimization.


1. Preparation of tissue.
2. SABC
1). Dewax:
Prepare three bottles of 90%, 95% and 100% dimethylbenzene and three bottles of 90%, 95% and 100% ethanol.
Immerse paraffin sections into three bottles of dimethylbenzene orderly (from low to high), 7min each. Then immerse into ethanol orderly at room temperature (from high to low), 7min each. Wash with water to remove ethanol.
Note: The process of dewaxing should be done in a fume hood at room temperature in summer. When the temperature is lower that 18¢J, it is recommended to dewax at 50oC.
2). Inactivation
Mix 30% H2O2 with distilled water(1:9). Immerse dewaxed paraffin section into the 3%H2O2 at room temperature for 10mim. Wash with distilled water for several times.
3). Repair
Heat repair: Immerse the paraffin sections into Citrate buffer (pH6.0), heat until boiling in the microwave and then cut off the power, keep it in the microwave for nearly 5˜10 mins. Repeat 1 or 2 times. Then cool at room temperature and wash 1 or 2 times with PBS(pH7.2-7.6).
3). Blocking
Add 5% BSA blocking solution or Normal goat serum and incubate at 37oC for 30min. Discard extra liquid, no washing.
4). Adding primary antibody
Dilute primary antibody: 1ug/ml is regarded as the concentration. Add some antibody diluents solution to primary antibody. Incubate at 37oC for 2 hours or overnight.
5). Wash with PBS for 2 times, 20min each. Add biotin-conjugated secondary antibody and incubate at 37oC for 30min.
6). Wash with PBS for 2 times, 20min each. Add SABC reagents and incubate at 37oC for 30min. Wash with PBS for 3 times, 20min each.
7). Add DAB reagents and incubate at RT. Wash with distilled water for several times.
8) Counterstain with haematoxylin. Dehydration and then immerse paraffin sections into dimethylbenzene twice, 7min each. Observe with microscope after seal


They have also provided new contact details to us from which we should receive much faster responses.

Thank you for your patience and understanding. I hope this will be helpful and I look forward to hearing from you with the results of the next experiments and details of how you would like to proceed.

Thank you for contacting us.
The antibody ab104686 is not in clear solution it is indeed in whole antiserum. We would recommend vortexing the antibody for a while and then after centrifuging using it as normal product. If in any case the antibody fails, please let me know we will replace the product.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
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Thank you for taking the time to complete our questionnaire. I am sorry to hear you have had difficulty submitting and sending the information to us. I can confirm we have now received the questionnaire and the images from you. I appreciate your patience.

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.

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

1. From the details provided,you have ordered through Lucerna Chem AG? I would appreciate if you could please check with them which order reference number they provided to us. We do have an order from them placed 31st January, PO B120092. Is this the correct one?

2. I can recommend to try different times for antigen retrieval as this can sometimes require some optimization. Try2, 5 and 10 minutes. andalso a sample withno antigen retrieval.

3. Could you confirm ifthe current vial of secondary antibody and the detection kit areworking well with other primary antibodies?

4. To help reduce non specific binding, I can suggest totry a lower antibody dilution 1:500 - 1:1000. Incubate overnight 4oC as this usually provides more specific and efficient staining.

5. I can recommend to wash the slides 3 times for 5 minutes in PBS containing 0.2%Tween. I can also suggest to include 0.2% Tween in the antibody dilution buffer. This will help to keep the antibody stabilized and also wash away any excess antibody.

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.

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

I would like to reassure you that this antibody is tested and covered by our 6 month guarantee forIHC-P in Mouse, Rat andHuman samples. In the event that a product is not functioning in the tested applications and species 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 would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily.


I would appreciate if you could also provide an image which would help us to assess the results.

Thank you for your time and cooperation. We look forward to receiving the completed questionaire.



Order Details
Antibody code: ab65854

Lot number:

Purchase order number
or preferably Abcam order number:


General Information
Antibody storage conditions (temperature/reconstitution etc)


Description of the problem (high background, low signal, non-specific satining etc.)

Sample (Species/Tissue/Cell Type/Cell Line etc.)


Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)


Antigen retrieval (Enzymatic method, Heat mediated technique etc.)
Borate buffer pH7 and with citrate buffer pH6 using the microwave at 98°C for 20 mins

Permeabilization step


Blocking conditions (Buffer/time period, Blocking agent etc.)


Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
1-2 µg/ml

Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Detection method


Positive and negative controls used (please specify)


Optimization attempts (problem solving)
How many times have you tried the IHC?



Have you run a "No Primary" control?
Yes No

Do you obtain the same results every time?
Yes No


What steps have you altered?


Additional Notes


We would appreciate if you are also able to provide and image which woudl help us to assess the results

Thank you for contacting Abcam regarding our CD40 and CD40L antibodies. Below please find the requested information: ab2391 Rabbit Polyclonal to CD40L - Unfortunately the specific immunogen sequence is proprietary, however I can inform you that it is a small peptide that surrounds aa246 of human CD40L. Also, I have learned from the laboratory that this antibody will react with mouse and is guaranteed to do so. ab58612 Rabbit Polyclonal to CD40 - The specific sequence is proprietary. Unfortunately, the blocking peptide is not commercially available. ab65854 Rabbit Polyclonal to CD40L - Please contact us for additional immunogen information. ab99894 Hamster monoclonal to CD40L - Unfortunately the epitope has not been mapped for this antibody. Regarding a possible negative control - for CD40 and CD40L I would recommend liver. I hope this information is helpful. Please do not hesitate to contact me if you have any additional questions.

Thank you for your enquiry. Please find the protocols as below: 1.Cover the entire surface of a clean microslide with APES or POLY-L-LYSINE. Incubate for 1 minute then rinse the microslide with water. Mount a tissue section (~5μm thick) with the treated microslide and bake in an oven at 58-60 ˚C for 30-60 minutes to ensure strong adhesion of the tissue section. 2.Dewax the tissue section in dimethylbenzene for 10 minutes and rinse with water. 3.Incubate the tissue section for 5~10 minutes in the 3% H2O2 solution to quench the endogenous peroxidase activity. Wash the tissue section with distilled water 3 times for 2 minutes each. 4.To heat repair the antigen, soak the tissue section in 0.01M citrate buffer (pH6.0), and heat to the boiling point with an electric heater or a microwave oven, then stop heating. Repeat this heating process 1~2 times with a 5~10-minute interval if necessary. Wash the tissue section with 0.02 M PBS (pH 7.2~7.6) once or twice when it cools to room temperature. 5.Add 5% BSA blocking reagent solution to the tissue section and incubate at room temperature for 20 minutes. Discard the blocking reagent solution, but do not wash the tissue section. 6.Add properly diluted primary antibody (mouse/rabbit IgG) to the tissue section and incubate at 37 ˚C for about 1 hour or 20 ˚C for about 2 hours or at 4 ˚C overnight. Wash the tissue section with 0.02M PBS (pH 7.2~7.6) 3 times for 2 minutes each. (The primary antibody concentration, incubation time and temperature directly affect the staining efficiency and background intensity. If the positive staining is too weak, the concentration of the primary antibody and the incubation time can be increased; if the background is too high, the primary antibody concentration and the incubation time can be decreased. ) 7.Add secondary antibody (20ug/ml) to the tissue section and incubate at 20~37˚C for 20 minutes. Wash the tissue section with 0.02M PBS (pH 7.2~7.6) 3 times for 2 minutes each. 8.Add SABC-Peroxidase (Streptavidin-Peroxidase) to the tissue section and incubate at 20~37˚C for 20 minutes. Wash the tissue section 4 times with 0.02M PBS (pH 7.2~7.6) for 5 minutes each. 9.Use a DAB chromogenic kit to stain the tissue section. Add Reagent A, B and C, one drop each, into 1 ml of distilled water and mix thoroughly. Add this solution to the tissue section and incubate at room temperature. Control the time of incubation under a microscope. Usually 5~30 minutes is sufficient. Wash the tissue section with distilled water. 10.Slightly counterstain the tissue section with haematoxylin, and wash with distilled water to clean the haematoxylin. Then dry the tissue section by baking, and put on a drop of resin seal the tissue section with a cover slide. The tissue section is ready for observation under a microscope.